Despite the overall decline in human immunodeficiency virus (HIV) prevalence, it remains unacceptably high in key populations. This study aimed to estimate prevalence of HIV infection and high-risk behaviors among female sex workers (FSWs). This study was a cross-sectional survey of FSWs conducted between December 2019 and August 2020 in eight geographically diverse cities in Iran. After providing informed consent, participants completed a bio-behavioral questionnaire and after completion of the interview, rapid diagnostic tests (RDT) and dried blood spots (DBS) were collected to determine HIV and syphilis status as per national guidelines, if the women consented. All analyzes were based on RDS adjustment. Weighting was done according to Giles’SS estimator. Among the 1515 FSWs, the overall prevalence of HIV and syphilis were 1.6% (95% CI: 0.8–2.3) and zero, respectively. The highest HIV prevalence was observed in Shiraz (5.1%) and Khorramabad city (1.6%). In addition, HIV-positive FSWs had a higher mean age at first sexual contact than HIV-negative FSWs (21.4 ± 5.6 vs. 17.2 ± 4.2 years). Injection drug use and a history of arrest or incarceration were associated with an increased odds of HIV infection (OR = 6.25; 95% CI: 1.90-20.55 and OR = 4.57; 95% CI: 1.64–12.72, respectively). Based on the results, strategies to improve early HIV diagnosis, harm reduction, and use of testing and treatment strategies are needed to reduce and control HIV infections.