In August 2012, the Houston Department of Health contacted CDC regarding the rare transmission of human immunodeficiency virus (HIV) likely by sexual contact between two women. The case was investigated, and laboratory testing confirmed that the woman with newly diagnosed HIV infection had a virus virtually identical to that of her female partner, who was diagnosed previously with HIV and who had stopped receiving antiretroviral treatment in 2010. This report describes this case of HIV infection, likely acquired by female-to-female sexual transmission during the 6-month monogamous relationship of the HIV-discordant couple (one negative, one positive). The woman with newly acquired infection did not report any other recognized risk factors for HIV infection, and the viruses infecting the two women had ≥ 98% sequence identity in three genes. The couple had not received any preventive counseling before acquisition of the virus by the woman who had tested negative for HIV. HIV-discordant couples should receive counseling regarding safer sex practices, and HIV-infected partners should be linked to and retained in medical care.