The study's objectives were to investigate the prevalence, incidence, persistence, and associated risk factors of syphilis in female sex workers (FSWs) in Kaiyuan City, Yunnan, China. Three serial cross-sectional surveys were conducted and biological specimens were collected and tested for HIV, sexually transmitted infections, and drug use. The logistic Generalized Estimating Equation regression model was used to identify risk factors for prevalent syphilis. The prevalence of syphilis was 7·5%, 8·4% and 8·8%, respectively, in the three survey periods. Estimated syphilis incidence was 1·07 cases/100 person-years, and the persistence of syphilis per person at 6 months was 90·4%. In multivariate analysis, the factors associated with syphilis were age, lower education level, number of clients in a week, inconsistent condom use with clients, herpes simplex virus type 2 (HSV-2), and Chlamydia trachomatis. Persistent syphilis in this population of FSWs is a serious public health concern.