Several characters in With Pearl and Ruby Glowing are interpreted as transgender when they were not written as such in the original source material. In many cases, this contributed to their abuse. It's also a frequent means to explain male characters giving birth to children, or occasionally female characters fathering them.
- Mr. Willard Tweedy is now Ms. Wilhemina Tweedy, having come out as trans after leaving her abusive wife, who sexually assaulted her after learning she was trans and gave birth to the resulting child in secret after Wilhelmina ran away.
- Julien is a transgender man, playing off the fact that lemurs are matriarchal in real life. He was raped by a religious cult due to being trans, impregnating him with Mort, who is his child in this universe.
- Riley is nonbinary in this universe, though they aren't out to their classmates. This is based on a popular headcanon meant to explain Riley having mixed-gender emotions. Ratigan exploited their insecurity about their gender to assault them.
- Dr. Flug is a trans man, with 5.0.5. being his human son conceived after Ratigan raped him.
- Kenai is a trans man; he was taken by the Ark and forced to have sex with Koda.
- Ruby and Sapphire, in canon, are both genderless female-presenting aliens. In this fic, they are a cis woman and a trans woman, though it's kept ambiguous which woman is which; this allows Garnet to be their biological child. The other Gems appear to all be female-identified. Rose, Lapis, Peridot, and Ocean Jasper are confirmed DFAB by having given birth; Peridot is Lapis' mother, Rose died in childbirth with Steven, Lapis gave birth to Malachite, and Ocean Jasper gave birth to Zebra Jasper. Malachite and Zebra Jasper are DFAB babies, not old enough to state whether their identities may be different. Jasper is Malachite's and Zebra Jasper's intersex father, and she confirms at least one or two of the Famethyst have the same condition, specifically including Lace Amethyst. It's speculated but not confirmed that said condition is PAIS, which can cause a feminine appearance with fertile sperm. Holly Blue is the birth mother of Jasper and the Famethyst. Emerald is nonbinary and DMAB (Spinel, who was raped by them, reports that Emerald has a penis), and works as a drag queen like their voice actor. Amethyst is genderfluid, saying that she's a demigirl and sometimes a demiboy. Nice Lapis is a trans girl.
- Mew and Mewtwo are respectively a cis girl and cis boy instead of being genderless. Meowth is a trans girl, in honour of the canon Meowth's voice actor Maddie Blaustein. Bea is nonbinary and uses it/its pronouns as a Shout-Out to a mistranslated wiki. Sylveon is a human trans woman who never got to transition officially but explained her identity in letters to her cis sister Eevee. Hilda and Hilbert are a Composite Character trans girl, with Hilda being her chosen name and Hilbert her deadname, while Nate and Rosa are similarly composited into a cis boy who gets Dragged into Drag. Ash's Pikachu is a female pet chinchilla.
- Pleakley is a trans woman as opposed to a Wholesome Crossdresser.
- Lynn Loud Jr. is a trans girl, but Foxy Loxy and her friends have parents that are members of God's Will First, so they assault her for it. Lana is questioning her gender.
- Flynn Rider is a trans man; the corrupt cops that assaulted him called him "Princess" because of this.
- Francis is trans and was subject to transphobic remarks from the flies at the bar because his face still looked lady-like.
- Peekablue is a trans boy and was attacked by God's Will First after Flutterina encourages Glimmer to make a False Rape Accusation against him, unaware that he was trans.
- Muscle Man is trans according to the "Parks and Rec" chapter, which mentions that his chest is unbound.
- All of JNPR are trans, with Jaune and Ren being trans boys and Nora and Pyrrha being trans girls. Also, Penny Polendina and Ruby Rose are trans girls.
- Auntie Uncle is non-binary rather than "Uncle" being a first name, and uses the Spivak ey/em/eir pronouns.
- Zoisite is a trans girl as a reference to her She's a Man in Japan status.
- Mr. Baldwin is trans and pregnant (willingly, unlike other cases in the fic) as a reference to him being a male seahorse in the show.
- Elina is a trans woman, and Erika Juno's gender is described as "none gender with left fluid
". Laverna pretends to be trans online, but isn't really. - Steele is a trans man who gets pregnant after drunkenly raping Balto.
- Shaun is a trans boy. In "Coming Out", he came out to a still closeted Wilhelmina, who told him that he could stay with her and Wallace if his family didn't accept him.
- Stacie is a trans girl, being a Composite Character of Todd, Tutti, and her canon self.
- Zipp Storm is trans due to her Tomboyish Voice in canon, as well as for plot reasons.
- Perky, who was in canon a female peacock, is a hijra/trans woman here.
- Daffy Duck is depicted as very apathetic towards gender in general and testing out different pronouns.
- Pinocchio, Emira, and Meilin Lee are cisgender intersex. Pinocchio has genital agenesis and his assigned gender was determined through a blood test, while Emira has Turner Syndrome, giving her a DFAB appearance. Meilin is undergoing contrasexual puberty as an analogue to her canon panda transformation, and is keeping her beard.
- Beth Lestrade is a trans woman.
- Bidoof's trainer is nonbinary, using xe/xem/xir pronouns. Bidoof was female in the original short, judging by her tail shape, and is a girl here too, but Lucario doesn't have dimorphic traits in the cartoon, while here he's confirmed as a boy.
- Michael Collins uses he/she/they pronouns; it's not yet revealed what their specific identity is.
- Kanamori is canonically DMAB femme, but a specific identity and English pronouns haven't been stated officially; here they're a she/they trans woman.
- Angel Dust is a cis man, but with what he describes as a hormone problem which gives him breasts instead of his canonical puffy chest fur.
- Monkie Kid is canonically trans; here he's specifically a demiboy, using he/they pronouns. Red Son is canonically nonbinary but uses he/him pronouns in the show and the Mandarin pronouns ta/ta de in the fic.
- Lake is confirmed to be nonbinary and use co/cos pronouns.
- Lenore is a trans man who goes by Lenny.
- Natsuki is a trans girl, and according to Monika she has been molested by her father ever since she came out.
- Homestar and Marzipan are both trans, and Marzipan knocked up Homestar. The Cheat is non-binary and uses they/them pronouns whereas in the original source material, The Cheat's pronouns are he/him.
- Donny is a trans boy, as an analogue for his desire to be a unicorn. Bliss identifies as female as in canon and is able to get pregnant, but is intersex with ovotesticular disorder, was assigned male at birth, and chose to keep her masculine first name, Biswanath. Indian culture classifies her as hijra. Snake is nonbinary.
- Rainbow Mane is a trans woman, in reference to how she was voiced by a male actor.
- Officer Fox is a trans woman as a reference to how she was originally male in the manhwa Brave Hedgehog.
- The boy and girl from Skies Forever Blue —here named Taffy and Skye respectively— are trans, their deadnames being Hana and Toby as a reference to the video’s creators.
- Harry Potter is a trans boy, which lead to his transphobic teacher Umbridge forcing him to carve lines into the back of his hand. Hermione has also been confirmed to be a trans girl and the Weasleys all have a hereditary intersex condition. Dumbledore is a drag queen.
- Glinda is either a trans woman or a drag queen or somewhere in between - the distinction wasn't made as clear in 1969, when the scene featuring her is set.
- Morticia and Gomez are both trans; we see they received a card at Wednesday's birth reading IT'S PROBABLY A GIRL.
- Nimona is non-binary and goes by ne/nir/nem pronouns whereas in the original movie and graphic novel, Nimona's pronouns are she/her.
- Yasuho Hirose, a Composite Character with Koichi, is a trans woman. Jolyne Cujoh is a trans man and is now named Joel. Dragona Joestar is non-binary.
- Sailor Uranus is canonically described as a blend of man and woman; here, he's specifically a bigender he/him lesbian.
- Dolly and Dizzy are dilbaa - a Navajo-specific identity describing AFAB people with a masculine gender expression. By Western standards, they're butch/tomboyish cis girls, though the term can also cover trans men.
- Miss Tarantula is a trans woman which got her sent to a male prison when the gang was arrested.
- Talulah Robinson is a trans woman.
- Averted with Wolfie, who still identifies as male even though he prefers to wear traditionally feminine clothes.
- Smurfette is a trans woman.
- Little Rural Riding Hood was a Llamana, a Zuni-specific term for transfeminine people.
- Bluey, Bingo, and Muffin identify as brotherboys, an Australian Aboriginal-specific term for DFAB people with masculine identities. Muffin specifically appears to identify as female in most ways except for wanting her penis back, as she's intersex and was operated on in infancy. Alfie is mentioned to be a sistergirl, the analogous term for feminine DMAB people.
- Iris is a trans girl.
