Objective To describe the characteristics of female sex workers (FSW) in 12 Brazilian cities. Methods This was a cross-sectional 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.