Objectives The prevalence of syphilis, caused by the spirochete Treponema pallidum subsp . pallidum (TPA), remains high despite the availability of effective antibiotics. In the Netherlands, most syphilis cases are found among men who have sex with men (MSM). We studied the distribution of TPA strain types by molecular characterisation and related this to available characteristics. In addition, resistance to macrolides was assessed. Methods TPA DNA was extracted from 136 genital ulcer swab or skin lesions samples deriving from 135 patients diagnosed with syphilis in 2016 and 2017 at the Public Health Service in Amsterdam, the Netherlands. Molecular typing was done according to the enhanced CDC method (E-CDC), in which three genetic regions of the arp , tpr and tp0548 genes are analysed by gel electrophoresis of the arp and tpr regions and by sequence analysis for the tp0548 region. Part of the 23S rDNA locus was sequenced to determine the presence of macrolide resistance-associated mutations. Results Full E-CDC strain types could be determined for 99/136 (73%) DNA samples, which tested positive in a diagnostic PCR targeting the polA gene. Types differed within one patient of whom two samples were available. No association was found between the demographic and clinical characteristics and the TPA types. The most prevalent type was 14d/g, found in 23 of the 99 (23%) fully typed samples. Part of the 23S rDNA locus was successfully sequenced for 93/136 (68%) samples and 83 (88%) contained the A2058G mutation. No A2059G mutation was found. Conclusions A broad strain distribution was found. Few subtypes were clonally expanded, and most other subtypes were rare. Detection of the most prevalent strain type, 14d/g, is in concordance with other TPA typing studies. The high prevalence of genetic macrolide resistance indicates that azithromycin is not an alternative treatment option.