Despite interest in the origins of syphilis, paleopathological analysis has not provided answers, and paleogenetic diagnosis remains a challenge. Even venereal syphilis has low infectivity which means there are few circulating bacteria for most of the individual’s life. Human remains recovered from the Nossa Senhora do Carmo Church (17th to 19th centuries) and the Praça XV Cemetery (18th to 19th centuries), Rio de Janeiro, Brazil, were subjected toTreponemapaleogenetic analysis. Historical data point to endemic treponemal infections in the city, including venereal syphilis. Based on the physiopathology ofTreponema palliduminfection, 25 samples, mostly from skull remains of young adults, with no visible paleopathological evidence of treponematoses, were analyzed. PCR with three molecular targets,tpp47,polA, andtpp15, were applied. Ancient DNAtpp15 sequences were recovered from two young adults from each archaeological site and revealed the polymorphism that characterizesT. p.subsp.pallidumin a female up to 18 years old, suggesting a probable case of syphilis infection. The results indicated that the epidemiological context and the physiopathology of the disease should be considered in syphilis paleogenetic detection. The findings ofTreponemasp. aDNA are consistent with historical documents that describe venereal syphilis and yaws as endemic diseases in Rio de Janeiro. Data on the epidemiological characteristics of the disease and its pathophysiology offer new perspectives in paleopathology.