I have seen a post going round tumblr that claims there are only four canon lesbian characters in video games. While games clearly need more lesbian characters, there are far more than four lesbian characters, they are just quite hard to find unless you know where to look. This is an attempt to rectify this.
Here’s every lesbian character (major and minor) I was able to find in chronological order. This does not include characters you can create yourself as that is optional role-playing. As usual, all corrections and suggestions are welcome.
It Came From the Desert (1989) - minor character, couldn’t find her name (this game is set in 1951).
Laura Bow in the Dagger of Amon Ra (1992) - two lesbian characters having an affair, couldn’t find their names (this game is set in the 1920s).
The Last Express (1997) - Sophie de Bretheuil and Rebecca Norton are a lesbian couple.
Gabriel Knight 3: Blood of the Sacred, Blood of the Damned (1999) - Lady Howard and Estelle Stiles are a lesbian couple.
The Longest Journey (1999) - Fiona and Mickey (who are the protagonist’s landladies) are a lesbian couple with a large age difference who have been together for at least 15 years.
Evil Zone (1999) - This fighting game has 3 lesbian characters: Setsuna Saizuki, Kakurine and Ihadurca,
Fear Effect 2: Retro Helix (2001) - This game featured a lesbian romance between two of the three protagonists, however this was apparently planned to develop into bisexual love triangle in a sequel that was ultimately cancelled.
Star Wars: Knights of the Old Republic (2003) - Juhani is a Cathar Jedi party member. She is a lesbian character, however a bug in early releases of the game made her occasionally available to a male player character. The game also implies that Juhani and another female Jedi, Belaya, were also lovers and if Juhani is killed by the player in their first meeting, Belaya will become a Sith to avenge her.
Singles: Flirt Up Your Life (2003) - Red is a lesbian option.
Buffy the Vampire Slayer: Chaos Bleeds (2003) - Features Tara Maclay and Willow Rosenburg (though whether Willow is lesbian or bisexual is open to debate).
CSI: Dark Motives (2004) - One of the murder cases in this game revolves around a pair of actress who were having an affair.
Fire Emblem: Radiant Dawn (2007) - Heather can be romanced by female soldiers.
Fatal Hearts (2007) - This visual novel dating sim has a lesbian option in Lucy Torville.
Fable II (2008) - This game allows sex and marriage with NPCs of lots of differing sexual orientations including lesbian ones.
Fallout 3 (2008) - Carol and Greta are two female ghouls (humans who have been exposed to large amounts of radiation and become disfigured) in a lesbian relationship.
Valkyria Chronicles (2008) - Dallas is a lesbian.
Heileen (2008) - This visual novel includes three love interests, one of which is a lesbian (the other two are a bisexual woman and a straight man).
Glory of Herakles (2010) - One of the protagonists, Leucos, is a cross-dressing lesbian, however this is played as a source of humour.
Scott Pilgrim vs. the World: The Game (2010) - Roxie Richter is a lesbian.
Fallout: New Vegas - Betsy, Veronica Renata Santangelo, and Christine Royce are all lesbians.
Dead Rising 2 (2010) - Two of the villains are implied to be in a lesbian relationship, however since they are also twin sisters this is likely played for shock value.
Fable III (2010) - Again allows sex and marriage with NPCs of lots of differing sexual orientations including lesbian ones.
Magical Diary (2011) - Virginia Danson is implied to be a lesbian (the other female romance options are implied to be bisexual).
Don’t take it personally babe, it just aint your story (2011) - Kendall and Charlotte are lesbians.
Love and Order (2011) - This visual novel has a lesbian option in Dorothy.
The Hookup (2011?) - This flash game features a lesbian option in Sara.
Star Wars: Knights of the Old Republic (2011) - A lesbian romance option was added in the Rise of the Hutt Cartel expansion pack.
Spirited Heart Girls’ Love/Spirited Heart Deluxe (2011?) - This life sim comes with four female love interests.
Analogue: A Hate Story (2012) - This visual novel features two minor characters who have a doomed lesbian affair.
Heileen 3: Sea Maidens (2012) - This conclusion of the Heileen trilogy has four female romances, however only Marie is a lesbian.
The Secret World (2012) - This MMO features seveal minor lesbian characters.
Guild Wars 2 (2012) - Caithe and Faolain are in a lesbian relationship and Kasmeer Meade and Marjory Delaqua are also in a lesbian relationship.
Borderlands 2 (2012) - In audio logs it is revealed that two extremely minor female characters are married.
Danganronpa 2: Goodbye Despair (2012) -Tsumiki Mikan and Junko Enoshima are in love.
Planet Stronghold (2012) - This sci-fi rpg/visual novel features a lesbian romance option in Michelle Lafleur.
Mass Effect 3 (2012) - Samantha Traynor is a lesbian romance option.
Zombies, Run! (2013) - This running app features a lesbian couple: Doctor Maxine Myers and Doctor Paula Cohen.
Gone Home (2013) - This game focuses on the lesbian relationship between the protagonist’s younger sister Samantha and her girlfriend Lonnie.
+Hate Plus (2013) - This sequel to Analogue: A Hate Story again features lesbian characters.
The Last of Us (2013) - Ellie has been confirmed by the creators to be a lesbian, as seen in the DLC Left Behind where she kisses Riley.
Baldur’s Gate II: Enhanced Edition (2013) - This version of the game adds an evil vampire lesbian romance option called Hexxat.
Borderlands: The Pre-Sequel (2013) - Janey Springs is a lesbian and Athena becomes her love.
Dragon Age: Inquisition (2014) - Sera is a lesbian romance option.
Seasons of the Wolf (2014) - Chalassa is a lesbian romance option.
Enola (2014) - The main character is a lesbian
Belladonna (2015) - The main character is a lesbian
Asphyxia (2015) - The main character is a lesbian
Brilliant Shadows - Part One of the Book of Gray Magic (2015) - Main character is a lesbian, several other lesbian characters.
Overwatch (2016) - Blizzard recently revealed that Tracer/Lena Oxton has a girlfriend and shortly after confirmed that she is a lesbian.