Background Sexually transmissible infection (STI) history, prevalence and seroprevalence among lifetime exclusive women who have sex with women (WSW) and an age-matched group of women who have sex with women and men (WSWM) was evaluated. Methods: Participants completed a study questionnaire and had genital specimens and sera collected for STI testing. Results: Twenty-one lifetime exclusive WSW and 42 WSWM were included. WSWM were more likely to report a history of prior STIs and be seropositive for chlamydia and HSV-2. Prevalent STIs were less common among WSW. Conclusions: While lifetime exclusive WSW are at risk of contracting STIs, WSWM are disproportionally affected. Healthcare providers should consider routine STI screening among WSW.