The species Treponema pallidum includes three subspecies (pallidum, pertenue, and endemicum) that cause syphilis, yaws, and bejel, respectively. A closely related species, Treponema paraluiscuniculi, is the etiologic agent of venereal syphilis in rabbits but does not infect humans. Although these treponemes cause distinct diseases, no laboratory method for differentiation has been reported. Genetic signatures were defined in the 5′ and 3′ flanking regions of the 15-kDa lipoprotein gene (tppl5) that distinguish the human pathogens and T. paraluiscuniculi, as well as distinguishing T. pallidum subsp. pallidum from the causes of human nonvenereal treponematoses. A single Eco47III restriction site in the 5′ flanking region differentiates T. pallidum subsp. pallidum from the other subspecies and species, and an XcmI site in the 3′ flanking region differentiates T. paraluiscuniculi from the human pathogens. Polymerase chain reaction methods and restriction polymorphism were used to analyze 27 strains of pathogenic Treponema species.