Treponema pallidum subspecies pallidum causes syphilis, a complex multi-stage sexually transmitted infection. Despite the availability of an effective antibiotic, penicillin, its prevalence remains worldwide. In high-income countries, the vast majority of syphilis cases occur among men who have sex with men. This thesis focused on molecular syphilis diagnostics and mapping the molecular variation of Treponema pallidum in Amsterdam, the Netherlands among different populations and within patients. Part I “Molecular Diagnostics” describes two studies that found Treponema pallidum in different anatomical locations within patients with syphilis in different infectious syphilis stages. These different anatomical locations were explored for diagnostical purposes and, by adding an additional diagnostical assay to the routine syphilis diagnostics, allowed some timely syphilis diagnoses in a screening setting of a population at increased risk of acquiring STI. Part II “Molecular Epidemiology” describes five studies in which a variety of molecular typing techniques are used. The most prevalent strains found in Amsterdam were similar to other countries. Intra-patient homogeneity of Treponema pallidum strains was found. Lastly, the impact of the first COVID-19 lockdown in 2020 on the genetic diversity of Neisseria gonorrhoeae was investigated and showed a major genetic and phenotypic shift. The effect of public health measures on the epidemiology of other infectious diseases should be taken into consideration for surveillance. Sharing data publicly and encouraging (inter)national collaborations will improve research.