We evaluated a molecular subtyping system for Treponema pallidum for its ability to differentiate between strains obtained from male patients with primary syphilis in South Africa. Of 201 T. pallidum-positive specimens, 161 were typeable, revealing 35 subtypes. The unique subtypes identified in Durban, Cape Town, and Carletonville and the total number of subtypes suggested that the strain population was very diverse and varied geographically.