Gender in My Little Pony

For the most part, all biological life-forms in the known multiverse seem to fall into two sexes, males and females respectively, though artificial life forms such as Robots do not seem to factor into these categories. Although a purely biological concept, the idea of ones sex and gender carries on into the afterlife, with ghost obviously carrying their gender identities.

Homosexuality
While rare, romance between same-sex couples have been documented in the land of Equestria. The most prominent example of this was probably with and, an open and proud same sex couple, though whether or not they were married or long tme girlfriends was never brought up. Another major example was and, who proposed to each other during "The Big Mac Question" and got married a little before Twilight's coronation during "The Last Problem".

The character Sappho, if going by artwork by her creator, was most likely a lesbian, or at the very least, bisexual and had a crush on.