This study evaluated risk factors associated with prevalent HIV-1 infection among pregnant women in a semi-rural but densely populated area surrounding the town of Butare in Rwanda. Overall seroprevalence was 9.3% in 5690 pregnant women who sought antenatal care at one of five health centres. Factors associated with higher seroprevalence of HIV-1 included history of multiple sexual partners, history of at least one sexually transmitted disease (STD), relatively high socioeconomic status (SES), being unmarried, young age at first pregnancy, and low gravidity. Women who had used oral contraceptives, smoked more than one cigarette per day, whose partners were circumcised, and had had sex to support themselves were also at higher risk of being infected. A history of blood transfusion in the past 5 years was not associated with HIV-1 infection. History of multiple sexual partners, history of STD, high household income, partner circumcision, and past oral contraceptive use remained strongly associated with HIV-1 infection even when simultaneously controlling for other covariates. Among legally married women who lacked sexual behaviour risk factors, history of STD, high SES, young age at first pregnancy, and low gravidity were significantly associated with HIV-1 seroprevalence.