Recent comparative genomics studies have revealed genetic diversity among strains of Treponema pallidum, the causative agent of syphilis. Molecular tools based upon this genetic diversity have resulted in the development of methods for detecting T. pallidum in various body tissues and fluids, differentiating the T. pallidum subspecies, molecular typing of T. pallidum strains, and rapid detection of macrolide resistance in circulating T. pallidum strains. These tools have increased our understanding of the pathogenesis and epidemiology of syphilis. We will discuss our enhanced understanding of the efficacy of standard and alternative antibiotics for treatment of syphilis, new knowledge concerning the movement of strains within and between sexual networks, and the application of this knowledge to public health responses to syphilis outbreaks.