1 Nozomi Tojo (Toujou) - Love Live School Idol Project
2 Nanoha Takamachi - Magical Girl Lyrical Nanoha
She and Fate are the best written lesbian couple in all of fiction.
3 Sailor Uranus - Pretty Soldier Sailor Moon
My biggest childhood crush honestly
4 Fate Testarossa - Magical Girl Lyrical Nanoha
5 Cure Moonlight / Yuri Tsukikage - HeartCatch Precure
6 Neptune / Purple Heart - Hyperdimension Neptunia
7 Sailor Neptune - Pretty Soldier Sailor Moon
Neptune is actually bisexual.
8 Plutia / Iris Heart - Hyperdimension Neptunia
9 Yumi Yoshimura - Hi Hi Puffy Amiyumi
Yumi Yoshimura, also known as Sue, is a Japanese musician, singer, and voice actress who is a member of the Japanese pop duo PUFFY, along with Ami Onuki. Fun Fact: The real-life Ami and Yumi actually thought that the cartoon versions of themselves were lesbians due to their unusually close bond with each other and how they talk about being best buds very often. However, the real-life Ami and Yumi are straight.
10 Signum Yagami - Magical Girl Lyrical Nanoha
Signum is a female fictional character that made her debut in the 2005 Japanese animated series Magical Girl Lyrical Nanoha A's.
The Contenders
11 Tracer - Overwatch
Tracer is a playable character appearing in the 2016 video game Overwatch, a first-person shooter video game developed by Blizzard Entertainment. She is confirmed to be one, which surprised me.
Tracer is a girl? I thought he was a boy?
12 Korra - The Legend of Korra
Avatar Korra is the title lead character in Nickelodeon's animated television series The Legend of Korra, in which she is depicted as the current incarnation of the Avatar, responsible for maintaining peace and balance in the world. Korra being gay for Asami came out of nowhere.
Technically she's Bisexual.
I love The Legend Of Korra!
13 Tsubomi Hanasaki / Cure Blossom - HeartCatch Precure
According to the HeartCatch Precure manga, she states that she will always love Itsuki regardless of gender.
14 Shamal Yagami - Magical Girl Lyrical Nanoha
15 Tomoyo Daidouji - Cardcaptor Sakura
She blushes around Sakura.
16 Subaru Nakajima - Magical Girl Lyrical Nanoha
Remember: Rainbows are straighter than Subaru.
In all seriousness, if you've watched MGLN StrikerS, it's pretty obvious that she's lesbian for Teana.
17 Lammy - Um Jammer Lammy
Um Jammer Lammy is a rhythm video game developed by NanaOn-Sha and published by Sony for the PlayStation video game console in 1999. A disturbingly hot one too
18 Velvet Crowe - Tales of Berseria
19 Alphys - Undertale
Alphys is an adorably dorky, lab-coat-wearing, bespectacled, yellow-skinned lizard scientist who resides in her lab in Hotland in the critically acclaimed cult classic RPG known as Undertale, wherein she partakes in the daily act of watching insane amounts of anime and having a vast majority of the game's plot-related spoilers attached to her.
Plot twists aside, however, she is also well known for creating the fabulous entertainment robot Mettaton, as well as having a massive crush on a young fish woman named Undyne, thus creating one of the most beloved lesbian couples in fiction.
20 Undyne - Undertale
Undyne is a main character in Toby Fox's 2015 RPG, Undertale. She is the heroic fishlike captain of the Royal Guard, who takes it upon herself to protect the monsters of the Underground. Her name is derived from the word "Undine", and is a pun on the word "Undying". Her most loved monsters are her mentor, King Asgore, her best friend, Papyrus, and her love interest, Alphys.
21 Patty Bouvier - The Simpsons
22 Marisa Kirisame - Touhou
23 Ami Onuki - Hi Hi Puffy Amiyumi
24 Tenko Chabashira - Danganronpa V3
25 Seimy - Jūsō Kikō Dancouga Nova