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Human immunodeficiency virus (HIV) prevalence is often high among female sex workers (FSWs) in sub-Saharan Africa. Understanding the dynamics of HIV infection in this key population is critical to developing appropriate prevention strategies. We aimed to describe the prevalence and associated risk factors among a sample of FSWs in Rwanda from a survey conducted in 2010. A cross-sectional biological and behavioral survey was conducted among FSWs in Rwanda. Time–location sampling was used for participant recruitment from 4 to 18 February 2010. HIV testing was done using HIV rapid diagnostic tests (RDT) as per Rwandan national guidelines at the time of the survey. Elisa tests were simultaneously done on all samples tested HIV-positive on RDT. Proportions were used for sample description; multivariable logistic regression model was performed to analyze factors associated with HIV infection. Of 1338 women included in the study, 1112 consented to HIV testing, and the overall HIV prevalence was 51.0%. Sixty percent had been engaged in sex work for less than five years and 80% were street based. In multivariable logistic regression, HIV prevalence was higher in FSWs 25 years or older (adjusted odds ratio [aOR] = 1.83, 95% [confidence interval (CI): 1.42–2.37]), FSWs with consistent condom use in the last 30 days (aOR = 1.39, [95% CI: 1.05–1.82]), and FSWs experiencing at least one STI symptom in the last 12 months (aOR = 1.74 [95% CI: 1.34–2.26]). There was an inverse relationship between HIV prevalence and comprehensive HIV knowledge (aOR = 0.65, [95% CI: 0.48–0.88]). HIV prevalence was high among a sample of FSWs in Rwanda, and successful prevention strategies should focus on HIV education, treatment of sexually transmitted infections, and proper and consistent condom use using an outreach approach.