Objectives: To measure the prevalence of HIV and related risk behaviors among female sex workers (FSW) in Khartoum State in northern Sudan. Methods: 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. Results: 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. Conclusion: 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.