Despite the high prevalence of HIV and STIs among women in Africa and the growing literature on HIV and STIs among women who have sex with women, research on the sexual health of women who have sex with women in Africa is scant. This study used mixed methods to describe sexual identity, practices and health among women who have sex with women in Lesotho. Most respondents (48%) described themselves as lesbian, 29% as bisexual and 23% as heterosexual. Almost half (45%) had disclosed their same-sex attraction to family, but only 25% had done so with healthcare workers. A total of 8% reported having HIV. Self-reported HIV was associated with having three or more male partners, having male and female partners at the same time and having a history of STIs. Gender norms, the criminalisation of homosexuality, varied knowledge of, and access to, safer-sex strategies, and mixed experiences of HIV/STI testing and sexual healthcare provided social and structural contexts for HIV- and STI-related vulnerability.