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 Aunt Holiday and Aunt Lofty, 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 Lyra Heartstrings and Bon Bon, 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 Rarity.

Wiz Kid and Curly Winds are generally believed to be a same-sex couple too, given the amount of seemingly romantic interactions between him.

Sunset Shimmer was confirmed to be bisexual, and in older drafts of Equestria Girls: Rollercoaster of Friendship, Applejack and Rarity were to reveal their romantic feelings for one another, which was later poked fun at with the introduction of the characters Ragamuffin and Dirk Thistleweed, who resembled Applejack and Rarity respectively and served as pseudo-love interests for Rarity and Applejack respectively.

According to Big Jim, it was left up to the individual viewers to decide whether or not Rainbow Dash and Applejack got together during "The Last Problem".