Introduction: Female sex workers (FSWs) are considered a bridge for transmission of pathogens from high-risk to general populations. We assessed the epidemiological status of syphilis in FSWs along the Pará highway system. Methods: Two hundred and twenty-two FSWs were interviewed and samples were analyzed using rapid qualitative tests and real-time polymerase chain reaction. Results: The prevalence of syphilis was high (36.94%). The high rate of prostitution, use of illicit drugs, and search for financial resources increased Treponema pallidum transmission through unprotected sex. Conclusions: Several characteristics of FSWs were identified, which reinforce the need for measures guaranteeing their health and protection.