Over the past decade, the incidence of syphilis and widespread macrolide resistance in its etiological agent, Treponema pallidum subsp. pallidum, have become a major health concern across countries, including China. Regional trends in subtypes and antibiotic resistance can be monitored effectively by molecular surveillance programs. In this study, whole blood samples were used to assess circulating T. pallidum strains collected from various regions of Hunan, China, between 2013 and 2015.