Objective: We sought to determine the prevalence of infection with Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, syphilis, and HIV among African American women who have sex with women (AAWSW), and compare sociodemographics, sexual risk behavior characteristics, and STI diagnoses among women reporting sex exclusively with women (exclusive AAWSW) to women reporting sex with both women and men (AAWSWM) during the past 12 months. Methods: Eligible women presenting to the Mississippi State Department of Health STD Clinic between February 2009 and October 2010 were invited to participate. A survey on sociodemographics, sexual history, and sexual risk behavior characteristics was completed. Women were tested for the presence of C. trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, syphilis, and HIV. Results: A total of 196 African American women were enrolled; 56.6% of all women reported engaging in sexual activity exclusively with women (AAWSW) during the past 12 months and 40.8% reported engaging in sexual activity with both men and women (AAWSWM). As compared with exclusive AAWSW, AAWSWM were significantly more likely to report prior infection with C. trachomatis (35.0% vs. 13.5%, P < 0.001), prior infection with N. gonorrhoeae (28.75% vs. 2.7%, P < 0.001), and transactional sex (18.8% vs. 2.7%, P = 0.001). Additionally, 13.8% of AAWSWM reported having sex with a homosexual or bisexual man during the past 12 months. Trichomoniasis was diagnosed in 18.3% of all women, C. trachomatis in 11.0%, M. genitalium in 7.6%, and N. gonorrhoeae in 3.7%. There were no cases of syphilis or HIV. AAWSWM were significantly more likely to be diagnosed with trichomoniasis (25.0% vs. 13.5%, P = 0.04), C. trachomatis (22.5% vs. 2.7%, P < 0.001), N. gonorrhoeae (7.5% vs. 0.9%, P = 0.01), or any STI (47.5% vs. 18.3%, P < 0.001) than exclusive AAWSW. Conclusions: AAWSW in this study were at high risk for STI. AAWSWM, as a subgroup, may demonstrate heightened sexual risk-taking behaviors and higher STI rates compared with exclusive AAWSW. Sexual health services provided to AAWSW should take into account partner gender heterogeneity when counseling and screening for STI. © 2011 American Sexually Transmitted Diseases Association All rights reserved.