| Character | Title | Author | Identity | Year | Notes |
| Aim/the Messenger | Nona the Ninth | Tamsyn Muir | Ambiguous/non-binary | 2022 | Aim uses they/them pronouns that were "bestowed upon them" by their role as the Messenger, is referred to with she/her pronouns and as "sir" by multiple characters, and has been stated by Tamsyn Muir to "live with bestowed pronouns and is violently proud of them while at the same time quite likes experiencing what other pronouns mean." |
| Alan | Two Strand River | Keith Maillard | Genderfluid | 1976 | One of the earliest literary novels to star gender-fluid characters. |
| Alanna of Trebond | The Song of the Lioness | Tamora Pierce | Genderfluid | 1983–1988 | Though the book itself never mentions her to be genderfluid, Pierce said in December 2019 that "Alanna has always defied labels. She took the best bits of being a woman and a man, and created her own unique identity. I think the term is 'gender-fluid', though there wasn't a word for this when I was writing her." |
| Annabel | Annabel | Kathleen Winter | Genderfluid | 2010 | Born intersex and assigned male at birth, Wayne sometimes takes on the name "Annabel". |
| The Beetle | The Beetle | Richard Marsh | Genderfluid | 1897 | The villain of the novel, simply called the Beetle, is an Egyptian shapeshifter that appears as both male and female throughout the book. |
| Riley Cavanaugh | Symptoms of Being Human | Jeff Garvin | Genderfluid | 2016 | Riley writes a viral blog about being genderfluid, and struggles to come out to parents and friends, using they/them pronouns often. |
| Chick the Cherub | John Dough and the Cherub | L. Frank Baum | Ambiguous | 1906 | Chick the Cherub is a human child whose gender is ambiguous throughout the book. |
| Crona | Soul Eater | Atsushi Ohkubo | Unknown/Ambiguous | 2010 | Crona is a demon sword master, and they appear as an antagonist under the orders of Medusa for the first part of the series. Their gender is never mentioned throughout the series, however the author explicitly stated that their gender is unknown. In the Japanese text, they are referred to using gender-neutral pronouns. |
| Dust Devil | My Little Pony | Jeremy Whitley | Non-binary | 2020 | Dust Devil is a non-binary abada who uses the singular they/them pronouns. |
| Elliot | On a Sunbeam | Tillie Walden | Non-binary | 2018 | Elliot "Ell" is a non-speaking "mechanical genius" who uses they/them singular pronouns. |
| Magnus Chase & the Gods of Asgard | Rick Riordan | Genderfluid | 2015 | Introduced in the second MCGA book, The Hammer of Thor, Alex Fierro is described as "transgender and gender-fluid," going by both masculine and feminine pronouns depending on state of mind and even changing appearance to suit pronouns. |
| The Fool | Realm of the Elderlings Series | Robin Hobb | Genderfluid | 1995–2017 | There is a lot of debate among fans as to what The Fool's biological sex is, but in terms of gender/self identity the Fool canonically identifies as male and female at different times. |
| Jude Winters; Dallas; Stevie | Jude Saves the World | Ronnie Riley | Non-binary | 2023 | Jude is a twelve-year old middle-school student who has chosen this name to replace their deadname. Jude wants to be called they/them but is unable to ask their conservative grandparents to do so. Jude has two school friends: Dallas comes out to Jude as being gay while Stevie has a crush on another girl but feels she must hide her feelings. The book sparked political backlash in Texas in 2025. |
| Oscar François de Jarjayes | The Rose of Versailles | Riyoko Ikeda | Ambiguous | 1972–1973 | A young queer woman raised as a soldier, dressing and behaving as a man, and is regarded as having a masculine presence by other characters. She also sometimes refers to herself by gender neutral pronouns. She is uncomfortable about presenting feminine and wants to be considered as a "genderless god of war" rather than a "gendered" human being. |
| Inanna | The Wicked + The Divine | Kieron Gillen | Non-binary | 2014 | An incarnation of the Sumerian goddess Inanna in the 2014 Recurrence, formerly a teenager called Zahid who had tendency to "blend in". Inanna uses he/him pronouns, except for the last issue in which they use they/them pronouns. |
| Jan | Mission Child | Maureen F. McHugh | Non-binary | 1998 | A first-person narrator who was assigned female at birth and, over the course of the novel, lives first as a young woman, then as a man, and ultimately declares that "I am not man or woman, just Jan." |
| Kinetiq | Sovereign | April Daniels | Genderqueer | 2017 | Kinetiq is an Iranian-American genderqueer superhero who has light based superpowers. |
| Leslie | Two Strand River | Keith Maillard | Genderfluid | 1976 | One of the earliest literary novels to star gender-fluid characters. |
| Loki | Marvel Universe | Al Ewing | Genderfluid | 2014–present | Loki is a shapeshifter who takes on both male and female forms, alternating between using he/him and she/her pronouns, and does not feel like he has a gender or orientation. |
| Eleodie Maracavanya | Star Wars: Aftermath | Chuck Wendig | Non-binary | 2015–2017 | A pirate ruler referred to by either male, female or gender-neutral pronouns like "zhe" or "zher". |
| Mogumo | Love Me for Who I Am | Kata Konayama | Non-binary | 2018 | Mogumo is an AMAB non-binary high school student who generally presents femme, and is mistaken for a cross-dresser and invited to work at a cross-dresser maid cafe. |
| Najimi Osana | Komi Can't Communicate | Tomohito Oda | Ambiguous | 2016–present | Described as having an ambiguous/unknown gender/sex by official Japanese website. |
| Niamh | The Kaiju Preservation Society | John Scalzi | Non-binary | 2022 | Niamh is an Irish astronomer and physicist who travels to another dimension populated by kaiju monsters. Niamh is exclusively referred to with singular they pronouns. |
| Paul | Nona the Ninth | Tamsyn Muir | Ambiguous | 2022 | Paul is a fusion of the male Palamedes Sextus and the female Camilla Hect who is referred to with they/them pronouns and whose gender Tamsyn Muir has described as "a clusterfuck". |
| Porcelain | Secret Six | Gail Simone | Genderfluid | 2014–2016 | A new member of the Secret Six. When questioned about their gender presentation, replied "Some days I feel like a girl, other days, not-so-much." |
| Popeye the Sailor | Popeye | E. C. Segar; Doc Winner; Tom Sims; Bela Zaboly; Ralph Stein; Bud Sagendorf; Bobby London; Hy Eisman; R. K. Milholland | Amphibious | 1919–present | In a 1954 comic, Popeye says that he "wears both woman's an man's clothes" and refers to his gender as "amphibious". On International Non-Binary People's Day in 2021, the official Popeye Twitter account posted an image of said comic with the caption "It'sk Non-binary Day! Happy skelebration to all me amphibious pals!" In another older comic, Popeye describes himself as "both a mother an' father" to his son Swee'Pea." |
| Hero Shackleby | River of Teeth | Sarah Gailey | Non-binary or agender | 2017 | A poisons and demolitions expert and love interest of Houndstooth, Hero goes by singular they/them pronouns, and their gender assigned at birth is never mentioned. |
| Geoffrey Swivel | The Shepherd's Crown | Terry Pratchett | Agender | 2015 | Although raised as a boy, Geoffrey decides that they want to become a witch. When asked why they don't want to be a wizard, Geoffrey tells Tiffany Aching that they don't think of themself as a man, or anything really. They are "merely Geoffrey". |
| Geoffrey Swivel | Tiffany Aching's Guide to Being a Witch | Rhianna Pratchett, Gabrielle Kent | Agender | 2023 | Although raised as a boy, Geoffrey decides that they want to become a witch. When asked why they don't want to be a wizard, Geoffrey tells Tiffany Aching that they don't think of themself as a man, or anything really. They are "merely Geoffrey". |
| Travertine | On the Steel Breeze | Alastair Reynolds | Non-binary | 2013 | Travertine uses "ve/ver" pronouns, and there is no mention of it being unusual in the book. |
| Hange Zoe | Attack on Titan | Hajime Isayama | Non-binary/Androgynous | 2010 | They have an androgynous appearance in the manga and is referred to with they/them pronouns. |
| Character | Title | Portrayed by | Identity | Year | Notes |
| The Adjudicator | John Wick: Chapter 3 – Parabellum | Asia Kate Dillon | Non-binary | 2019 | The character did not have a specified gender in the script; Dillon, a non-binary person, came up with the idea of making their character - while talking with the director. |
| Alex | After the Hunt | Lío Mehiel | Non-binary | 2025 | Alex is Maggie's live-in partner. |
| All | Zoolander 2 | Benedict Cumberbatch | Androgyne | 2016 | All is presented as a famous androgyne supermodel. The character was subject to a large backlash, being described as "an over-the-top, cartoonish mockery of androgyne/trans/non-binary individuals." |
| A-Spen | Zombies 3 | Terry Hu | Non-binary | 2022 | An unambiguously non-binary space alien. There is a doll for A-Spen, and it will be the first non-binary doll from Disney. The pronouns on the box are they/them in multiple languages. |
| Biaggio | The Kings of Summer | Moisés Arias | Agender | 2013 | In the film, Biaggio states that he does not see himself as having a gender. |
| Bobo | One Battle After Another | Colton Gantt | Non-binary | 2025 | Bobo is one of Willa's friends. |
| Cass | Outerlands | Asia Kate Dillon | Non-binary | 2025 | Cass is a lonely nanny and restaurant server in San Francisco whose is confronted with her own childhood trauma when their coworker leaves Cass in charge of her 11-year old daughter. |
| Deadeye / Vanessa | Joy Ride | Sabrina Wu | Non-binary | 2021 | Deadeye is Lolo's eccentric cousin who is obsessed with K-pop. |
| Glen/Glenda | Seed of Chucky | Billy Boyd | Genderfluid | 2004 | Although Glen/Glenda was not explicitly identified as genderfluid in the movie, their genderfluid identity was confirmed in the Chucky TV series. |
| Fab G | Cinderella | Billy Porter | Genderless | 2021 | Fab G is the fairy godparent of Ella. |
| Frances | Jimpa | Aud Mason-Hyde | Non-binary | 2025 | Frances travels to Amsterdam with their mother to visit their gay grandfather Jim, and wishes to stay with their grandfather for a year. |
| J | They | Rhys Fehrenbacher | Genderfluid | 2017 | J is a trans teen on puberty blockers that needs to decide their gender before meeting with a doctor. J says they feel male, female, or neither at various times. The actor, Fehrenbacher, was also undergoing gender transition at the time of filming. |
| Jamie | Upgrade | Kai Bradley | Non-gendered | 2018 | A hacker not identifying with any of the genders. Requests that the protagonist not ask their gender, and states that Jamie is not their name and that they do not have a name. |
| S. LaFontaine | The Carmilla Movie | K Alexander | Non-binary | 2017 | In this film, and the web series it serves as a sequel to, LaFontaine uses singular they/them pronouns. |
| Lake Ripple | Elemental | Kai Ava Hauser | Non-Binary | 2023 | Lake Ripple is Wade and Alan's sibling. Lake uses they/them and she/her pronouns. Lake is Pixar's first non-binary character. Lake is voiced by non-binary actor Kai Ava Hauser. |
| Layla | Layla | Bilal Hasna | Non-binary | 2024 | Layla is a British-Palestinian non-binary drag queen who falls in love with the marketing executive Max. |
| Little Horse | Little Big Man | Robert Little Star | Two-spirit | 1970 | Little Horse is a two-spirit Cheyenne indigenous person. |
| Hedwig Robinson | Hedwig and the Angry Inch | John Cameron Mitchell | Genderqueer | 2001 | Hedwig is described by her creator as "more than a woman or a man. She's a gender of one." |
| Frankie Stein | Monster High: The Movie | Ceci Balagot | Non-binary | 2022 | Frankie, the child of Frankenstein and of Dr. Stein is a non-binary monster, made of different male and female body parts, who prefers they/them pronouns. |
| Yivo | Futurama: The Beast with a Billion Backs | David Cross | Non-binary | 2008 | Yivo is a planet-sized alien with no determinable gender, using neopronouns. Yivo dates, then marries all people of the universe at once. Later, they break up. Afterwards, Yivo remains in a relationship with Colleen O'Hallahan. Some have said that Yivo may have been "the first non-binary character defined as such in animated history." |
| Character | Show title | Portrayed by | Identity | Year | Notes |
| Alex | The A List | Rosie Dwyer | Genderqueer | 2018–2021 | Uses singular they/them pronouns along with she/her pronouns in the show. |
| Yael Baron | Degrassi: Next Class | Jamie Bloch | Genderqueer | 2016–2017 | Assigned female at birth, Yael begins to question their identity starting in season 4, before realizing they are genderqueer and uses singular they/them pronouns. |
| Kai Bartley | Grey's Anatomy | E.R. Fightmaster | Non-binary | 2021–present | Uses singular they/them. E.R. Fightmaster is also non-binary and uses they/them pronouns. |
| Skye Beck | The Summer I Turned Pretty | Elsie Fisher | Non-binary | 2023 | Introduced in season 2, Skye is the non-binary cousin of the Fisher brothers and uses singular they. They are a socially awkward teenager that becomes close to their cousins when their mom, Julia, makes plans to sell the family’s beach house. They are an original character to the series, not appearing in the novels the television show is based on. |
| Bishop | Deputy | Bex Taylor-Klaus | Non-binary | 2020 | Bishop is considered the first non-binary character on broadcast television. |
| Cal Bowman | Sex Education | Dua Saleh | Non-binary | 2021–2023 | Cal is a non-binary student at Moordale Secondary School, introduced in season 3. The headmistress, Hope Haddon, tries to force Cal to wear the girls' uniform, but Cal repeatedly defies Hope by wearing the boys' uniform instead. Uses singular they/them pronouns. |
| Lindsay Brady | Good Trouble | River Butcher | Non-binary | 2019–2024 | Uses singular they/them pronouns. |
| Sah Brockner | Casualty | Arin Smethurst | Non-binary | 2021–present | Sah is a non-binary paramedic who uses singular they/them pronouns. |
| Miss Bruce | Star | Miss Lawrence | Genderfluid | 2017 | Bruce is a fierce genderfluid person who became a fan favorite for those who watched the show. |
| Carlos Cervantez | The Winchesters | Jojo Fleites | Non-binary | 2022–2023 | Cervantes is a demon hunter. |
| Chris | The Switch | Amy Fox | Non-binary | 2016 | Chris uses "zie/zir" pronouns in the show. |
| Danny the Street | Doom Patrol | | Genderqueer | 2019–2022 | Danny is a sentient street who communicates through writing on street signs. They are explicitly referred to as non-binary and genderqueer onscreen. |
| Des | Grown-ish | Egypt Franklin | Genderfluid, Non-binary | 2021–2024 | Des, a queer, genderfluid, and pansexual athlete is a possible love interest of the character Jazz. |
| Desire | The Sandman | Mason Alexander Park | Non-binary | 2022–2025 | Like their comics counterpart, Desire is androgynous in appearance and is referred to with gender-neutral language and they/them pronouns, as well as being played by a non-binary actor. |
| Che Diaz | And Just Like That... | Sara Ramirez | Non-binary | 2025 | Diaz is a stand-up comic and podcast host. |
| The Doctor | Doctor Who | Various | Genderfluid | 1963–1989, 1996, 2005-present | The Doctor presents variously as male and female throughout the series, being referred to by both male and female pronouns. In the 2023 specials the Doctor says that they use "the" as a pronoun. |
| Grencia Mars Elijah Guo Eckener | Cowboy Bebop | Mason Alexander Park | Non-binary | 2021 | Gren is a 29 year old non-binary individual who is connected to Spike and Vicious' past, and is overtly shown as non-binary, as confirmed by Netflix and their voice actor as part of promotional information for the series. Gren describes themselves as "I am both at once, and neither one" in the original anime series the live action is based on, Cowboy Bebop. |
| Brooke Hathaway | Hollyoaks | Tylan Grant | Non-binary | 2018–present | An autistic person who learns about non-binary identities from non-binary friend Ripley Lennox. Brooke feels that they relate to the identity. |
| Cassidy Geoffrey | Abbott Elementary | Sabrina Wu | Non-binary | 2021–present | Mx. Geoffrey is the new substitute teacher running Janine's classroom. |
| GJ | Top of the Lake | Holly Hunter | Ambiguous | 2013–2017 | GJ is an androgynous Swiss spiritual leader. |
| Jim Jimenez | Our Flag Means Death | Vico Ortiz | Non-binary, Bisexual | 2022–2023 | Jim uses they/them pronouns, and they are played by a non-binary actor. |
| Jerrie Kennedy | Big Sky | Jesse James Keitel | Non-binary, Transfeminine | 2020–2023 | Kennedy is one of the first non-binary actors to play a non-binary series regular on primetime television. |
| S. LaFontaine | Carmilla | K Alexander | Non-binary | 2014–2016 | Uses singular they/them pronouns. |
| Loki Laufeyson | Loki | Tom Hiddleston | Genderfluid | 2021–2023 | A character from the Marvel Cinematic Universe who first appeared in Thor. Like his comics counterpart and the Norse deity he was based upon, Loki's shapeshifting abilities allow him to change sex at will. In the show, Time Variance Authority paperwork lists Loki's sex as "fluid". |
| Ripley Lennox | Hollyoaks | Ki Griffin | Non-binary | 2020–present | Ripley runs a shop for second-hand clothes and befriends some of the show's younger characters like Peri Lomax and Romeo Nightingale, while also an established friend of Tom Cunningham. They later come out to their friends as non-binary. |
| Jordan Li | Gen V | London Thor and Derek Luh | Non-binary, Bigender | 2023–present | After being injected with Compound V, Jordan is able to switch sex at will.> |
| Sam Malloy | The Riches | Aidan Mitchell | Non-binary, Trans woman | 2007–2008 | Sam, the youngest Malloy child, is transgender and frequently dresses in feminine clothing. The idea for Sam's non-binary gender expression came about before Izzard, a gender non-conforming comedian, joined the show. Sam's gender expression is accepted and respected by the Malloy parents and siblings. |
| Mae Martin | Feel Good | Mae Martin | Non-binary | 2020–2021 | Mae comes out as non-binary in the second season. |
| Taylor Mason | Billions | Asia Kate Dillon | Non-binary | 2016–2023 | A non-binary person who uses singular they/them pronouns and has a storyline centered on a romantic relationship. |
| Sabi Mehboob | Sort Of | Bilal Baig | Genderfluid | 2021–2023 | They are openly gender fluid and uses they/them pronouns. |
| Mo | Zoey's Extraordinary Playlist | Alex Newell | Genderfluid | 2020–2021 | He is openly genderfluid, and generally uses he/him pronouns, but is open to the use of any pronouns. |
| Lucifer Morningstar | The Sandman | Gwendoline Christie | Ambiguous | 2022–2025 | The show's version of Lucifer is never referred to with any third-person pronouns and is androgynous in appearance. |
| Darcy Olsson | Heartstopper | Kizzy Edgell | Non-binary | 2022–present | Darcy began experimenting with singular they/them pronouns and came out as non-binary in season 3. |
| Pollution | Good Omens | Lourdes Faberes | Non-binary | 2019 | Uses singular they/them pronouns; described by book co-author and series writer Neil Gaiman as non-binary. |
| Joey Riverton | Good Trouble | Daisy Eagan | Non-binary | 2019–2024 | Joey comes out as non-binary to their girlfriend, Alice, and begins using they/them pronouns. |
| Sam | Vida | Michelle Badillo | Non-binary | 2018–2020 | Sam's gender identity was not revealed until their sex scene with Emma Hernandez. |
| Slug | Ironheart | Shea Couleé | Non-binary | 2025 | Slug is a hacker from Madripoor and member of Parker Robbins' gang. They are a former drag queen who wants to redistribute wealth from the privileged to the Chicago community, and uses non-binary pronouns. |
| Syd | One Day at a Time | Sheridan Pierce | Non-binary | 2017–2020 | Uses singular they/them pronouns. Syd is also the significant other of Elena Maria Alvarez Riera Calderón Leyte-Vidal Inclán, an activist and feminist teenage daughter of Penelope. |
| Adira Tal | Star Trek: Discovery | Blu del Barrio | Non-binary | 2020 | Adira, the first non-binary character in the Star Trek universe, is a highly intelligent character on the U.S.S. Discovery and unexpectedly becomes friends with Lt. Commander Paul Stamets and Dr. Hugh Culber. Adira is also an introvert who does not originally tell the crew they are non-binary, using she/her pronouns until episode 8 when Adira comes out as non-binary and asks to be referred to as "they or them". |
| Tam | Younger | Jesse James Keitel | Genderqueer | 2018 | Uses singular they/them pronouns. |
| Lommie Thorne | Nightflyers | Maya Eshet | Genderfluid | 2019 | Lommie is a gender-fluid cyber technician specialist who prefers to interface with computers more than humans. She uses she/her pronouns. |
| Xavin | Marvel's Runaways | Clarissa Thibeaux | Genderfluid | 2019 | Xavin is a blunt, romantic alien who possesses shapeshifting abilities. In the series, Xavin is referred to with both they/them and she/her pronouns. |
| Zoey | The Switch | Vincent Viezzer | Genderqueer | 2016 | Zoey is a feisty "transgender genderqueer" woman who is guarded by her neighbor, Detective Sandra McKay, a cisgender lesbian. |
| Character | Game | Voice actor | Identity | Year | Notes |
| Azul | Neo Cab | | Non-binary | 2019 | Referred to with they/them pronouns in game, and with a bionic prosthetic arm replacing their arm which was amputated after an accident. |
| Mizuki Akiyama | Hatsune Miku: Colorful Stage | Satou Hinata | Genderqueer | 2020 | In flashbacks, Mizuki is shown with a boy's school uniform and shorter hair. In the present they have long hair and a girl's uniform. When pronouns are needed, they/them pronouns are used, and their gender on their profile is listed as "?". |
| Ash | Wandersong | | Non-binary | 2018 | Referred to with they/them pronouns in game. The game creator later confirmed they are a non-binary character. |
| The Bard | Wandersong | John Robert Matz | Non-binary | 2018 | The Bard is referred to with singular they/them pronouns, but it is also mentioned that any pronouns are fine for them in a QA session. |
| Blanche | Pokémon Go | | Non-binary | 2016 | Blanche is referred to using singular they/them pronouns in English-language official media. |
| Bloodhound | Apex Legends | Allegra Clark | Non-binary | 2019 | Bloodhound is referred to as non-binary and uses singular they/them pronouns. |
| Bolt | Crypt of the NecroDancer | | Genderqueer | 2015 | According to Ted Martens, artist of this video game, Bolt does not identify as "fully as either male or female". |
| Company Commander | BattleTech | | Non-binary | 2018 | The Company Commander is the player character. During character creation, the player can define their name, callsign, and pronouns independently of the portrait or background, potentially making them non-binary. Most hired MechWarriors can also be customized in this way during the game. |
| Chaos | Hades | Peter Canavese | Non-binary | 2019 | Characters in-game refer to Primordial Chaos with they/them pronouns. Additionally, they were referred to with such pronouns on the official Chaos Update from Supergiant Games' Twitter. |
| Mihaly/Coach of "Rather Be" | Just Dance 2023 Edition | | Non-binary | 2022 | The coach is referred to with singular they/them pronouns by the official Just Dance Twitter account. The coach is also confirmed to be non-binary in a response to a fan tweet. |
| Astoria: Fate's Kiss | | Non-binary | 2015 | A boss, with whom characters can engage in a romance with, with gender identity that is accepted by other characters. Uses singular they/them pronouns. |
| Jordan "JD" Davies | Havenfall is For Lovers | | Non-binary | 2017 | Uses singular they/them pronouns and is non-binary. |
| Kris Dreemurr | Deltarune | | Unstated | 2018 | Kris is the main protagonist of Deltarune. They are exclusively referred to with singular they/them pronouns inside and outside of the game, and can be equipped with or present in both masculine and feminine attire. |
| Floofty Fizzlebean | Bugsnax | Casey Mongillo | Non-binary | 2020 | Floofty Fizzlebean is referred to with they/them pronouns and by another character as their "sibling" in game. Developers have confirmed that they are intended to be non-binary representation. |
| FL4K | Borderlands 3 | SungWon Cho | Non-binary | 2019 | Fl4k was confirmed non-binary before the game's release, and is referred to with singular they/them pronouns in-game. They also wear a non-binary pride flag pin. |
| Efrim Waite | Neurocracy | | Non-binary | 2019 | Waite is always referred as a single individual using they/them pronouns in the in-game encyclopaedia. |
| Cirava Hermod | Hiveswap | | Non-binary | 2017 | Cirava is referred to with singular they/them pronouns in all official media. |
| Jo | Angry Birds | | Non-binary | 2023 | Jo, a bird of paradise, was first teased on Angry Birds social media pages on June 3, 2023, for Pride Month. They were included in Angry Birds 2 as a limited-time spell called "Jo's Looove Spell Event". |
| Charun Krojib | Hiveswap | | Non-binary | 2017 | Charun is stated by What Pumpkin, the production team, to be non-binary, and is referred to with singular they/them pronouns in all official media. |
| Lorelei | Borderlands 3 | Ciarán Strange | Non-binary | 2019 | Lorelei is a soldier and former barista who is non-binary and considering gender transition. |
| Nights | Nights into Dreams | | Agender | 1996 | Franchise developer Takashi Iizuka is cited as saying Nights has no gender, and that players were free to apply their own thoughts on Nights' gender if they wanted to. Nights' voice in Journey of Dreams and Racing Transformed is feminine, but the games treat them as agender. |
| Nights | Nights: Journey of Dreams | Julissa Aguirre | Agender | 2007 | Franchise developer Takashi Iizuka is cited as saying Nights has no gender, and that players were free to apply their own thoughts on Nights' gender if they wanted to. Nights' voice in Journey of Dreams and Racing Transformed is feminine, but the games treat them as agender. |
| Nights | Sonic & All-Stars Racing Transformed | Julissa Aguirre | Agender | 2012 | Franchise developer Takashi Iizuka is cited as saying Nights has no gender, and that players were free to apply their own thoughts on Nights' gender if they wanted to. Nights' voice in Journey of Dreams and Racing Transformed is feminine, but the games treat them as agender. |
| Sackboy | LittleBigPlanet | | Non-binary | 2008 | While referred to with he/him pronouns, he is confirmed on social media as non-binary by developer Mark Hall. |
| Setsu | Gnosia | | Non-binary | 2019 | Refers to themselves as non-binary in the game. |
| Raqio | Gnosia | | Non-binary | 2019 | Raqio is referred to with singular they/them pronouns. |
| Testament | Guilty Gear | | Agender | 1998 | Was previously referred to with he/him pronouns in the US due to localization issues. As of 2021's Guilty Gear Strive, Testament is now referred to with singular they/them pronouns and is confirmed by the developers to be agender. |
| Dominique Pamplemousse | Dominique Pamplemousse in "It's All Over Once the Fat Lady Sings!" | Dietrich Squinkifer | Genderqueer | 2013 | Dominique has a masculine appearance and a feminine voice, leading characters to question their gender. When asked if they are male or female, Dominique replies "I'm a detective." The itch.io page for the second and final game in the series describes Dominique as "our favourite genderqueer private detective". |
| Steve | Minecraft | | Agender | 2009 | Markus Persson, the creator of Minecraft, originally envisioned Steve as a non-binary character because "gender isn't a gameplay element". The name "Steve" was initially given to the character as a joke, but Persson later expressed regret over the name because it was a male given name. |
| Character | Title | Author | Identity | Year | Notes |
| Tilly Birch | Questionable Content | Jeph Jacques | Non-binary | 2003–present | Uses singular they/them pronouns. Character first appeared in 2017. |
| Davepetasprite^2 | Homestuck | Andrew Hussie | Non-binary | 2009–2016 | A fusion of a male character and a female character, Davepetasprite^2 had a short crisis with regards to their gender identity, but quickly settled as non-binary. The character first appeared in 2015. |
| Eth | Eth's Skin | Sfé R. Monster | Gender-neutral | 2014–present | Using singular they/them pronouns in the webcomic, author Sfé Monster has stated that Eth presents and identifies as gender-neutral. |
| Parker Flores | Chroma Key | Brandon Dumas | Non-binary | 2018–2021 | Parker is in a group of Tokusatsu-inspired heroes. Parker is also non-binary, and some of the narrative conflict comes from characters misgendering them. |
| List of [Homestuck characters|Calliope] | The Homestuck Epilogues | Andrew Hussie | Non-binary | 2009–2019 | Comes out as non-binary in the "Meat" path of The Homestuck Epilogues and uses singular they/them pronouns. They first appeared in 2012. |
| Roxy Lalonde | The Homestuck Epilogues | Andrew Hussie | Non-binary | 2009–2019 | Comes out as non-binary in the "Meat" path of The Homestuck Epilogues, initially using singular they/them pronouns and later masculine pronouns; in the "Candy" path of The Homestuck Epilogues, Roxy questions her gender, but ultimately continues to identify as female. Character first appeared in 2011. |
| Never Satisfied | Taylor Robin | Non-binary | 2015–present | Lucy is a non-binary magic apprentice out to prove themselves to an "apathetic master". They use singular they/them pronouns. |
| Patrick | Strong Female Protagonist | Brennan Lee Mulligan | Genderqueer | 2012–present | Patrick does not identify as a person, although primarily uses masculine pronouns. Patrick first appeared in 2012. |
| Patrick | Strong Female Protagonist | Lee Knox Ostertag | Genderqueer | 2012–present | Patrick does not identify as a person, although primarily uses masculine pronouns. Patrick first appeared in 2012. |
| Never Satisfied | Taylor Robin | Non-binary | 2015–present | A magic apprentice, who is non-binary. Rascal uses singular they/them pronouns. |
| Paranatural | Zack Morrison | Non-binary | 2010–present | R.J. uses singular they/them pronouns. |
| Serious Trans Vibes | Sophie Labelle | Genderfluid | 2014–present | Ciel is a non-binary girl and seventh grader. Ciel is one of the webcomic's protagonists and is dating Eirikur. |
| Never Satisfied | Taylor Robin | Non-binary | 2015–present | A magic apprentice, who is non-binary. Tetsu uses singular they/them pronouns. |
| The Order of the Stick | Rich Burlew | Genderqueer | 2003–present | Vaarsuvius' gender is deliberately ambiguous, and Rich Burlew has stated that descriptions of them as male or female by other characters do not reflect their gender. |
| Go Get a Roomie! | Chloé C | Agender | 2010–present | Watch is comfortable with whichever pronouns the speaker chooses and does not identify with any particular gender. |