Syphilis is a sexually-transmitted bacterial infection that re-emerged in the early 2000s. The treatment of syphilis is easy, cheap, and well tolerated, but its diagnosis remains challenging. In particular, it may be missed in the early phase of the disease and then evolve to more advanced stages, or provide a portal of entry for human immunodeficiency virus. Since the early 1990s, Treponema pallidum polymerase chain reaction (Tp-PCR) has been investigated as a complementary diagnostic tool. We first assessed the diagnostic performance of Tp-PCR in various biological specimens and syphilis stages and demonstrated Tp-PCR performed better in ulcers or skin lesions. We secondly conducted a multicentre prospective clinical study to evaluate the accuracy of Tp-PCR compared to darkfield microscopy in ulcers and we concluded that Tp-PCR could even replace darkfield microscopy in routine screening. Finally, we showed that the two current genes targeted for Tp-PCR could be univocally used.