Given reports of increasing syphilis incidence in Western countries, we used molecular typing and epidemiological data to elucidate Treponema pallidum transmission networks. Samples and data were collected, dating from 2002 to 2005, from a well-defined population of patients with an ulcus and a diagnosis of infectious syphilis. Molecular typing using the tprK gene (V3-V5 region) was performed on 211 isolates from 205 Amsterdam STI clinic patients. We revealed 32 T. pallidum clusters and recognized ten large clusters, consisting predominantly of homosexual men (89%). Yet, no common patient characteristics were found to link the patients in these clusters. We therefore conclude that the highly variable tprK region (V3-V5) is not suitable for elucidating T. pallidum transmission networks in a high risk population.