Objective: To describe the characteristics of female sex workers (FSW) in 12 Brazilian cities. Methods: This was a crosssectional biological and behavioral surveillance study with FSW recruited by respondent-driven sampling (RDS) in 2016. HIV prevalence, sociodemographic, behavioral and sex work profession variables were estimated by city. The chi-square test was used to compare these distributions. Results: Among 4,328 FSW, HIV prevalence was 5.3% (95%CI 4.5;6.3), ranging from 0.2% (95% CI 0.0;1.2), in Campo Grande, to 18.2 (95%CI 13.2;24.7) in Salvador. In half of the cities, HIV prevalence in the samples was >5.0%, while Campo Grande, Brasília and Belo Horizonte had prevalence rates <1.0%. Significant differences between cities were found in distributions according to educational level, income, workplace, age sex work started, and illicit drug use. Conclusion: The characteristics of the samples in each city were distinct and have influenced local HIV prevalence.