The Rainbow Alliance filed the complaint on Wednesday with the city's Commission on Human Relations.
The group represents gay, lesbian, bisexual and transgender people at the school.
It says the Pitt policy requiring transgender students to use bathrooms, locker rooms and other facilities that match the sex listed on their birth certificate is "extremely regressive" and unsafe, putting such students at increased risk of violence and harassment.
Pitt spokesman Robert Hill says the school does not comment on litigation, but does not discriminate based on gender identity and expression.
City officials say the complaint could take six to nine months to investigate.