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To measure the prevalence of HIV and related risk behaviors among female sex workers (FSW) in Khartoum State in northern Sudan.We conducted a cross-sectional survey using respondent-driven sampling (RDS) that included 321 FSW in Khartoum from April to May 2008. A face-to-face interview was completed using a standardized questionnaire and blood was collected for HIV testing. Population point estimates and 95% confidence intervals (CI) were generated using RDSAT to adjust for the peer-referral recruitment patterns of RDS.HIV prevalence was 0.9% (95% CI 0.1–2.2). The majority (69.1%) had multiple clients in their last working day and 71.3% were married or cohabitating. A condom was used by 45.0% of respondents at their last sex with a client and consistently in the last month by 35.9%. Comprehensive HIV/AIDS knowledge was demonstrated by 25.4% of FSW. Only 7% of respondents had sought voluntary HIV counseling and testing in the preceding 12 months.For the present, HIV prevalence among FSW in Khartoum appears lower than the previous estimates. However, conditions may foster wider transmission in the absence of concerted interventions.