The aim of this nationwide study is to determine the strain type diversity among patients diagnosed with syphilis by PCR during a 4-year period in Denmark. Epidemiological data, including HIV status, for all patients were obtained from the Danish national syphilis registration system. Molecular strain typing was based on characterization of 3 variable treponemal genes, arp, tpr and tp0548. A total of 278 specimens from 269 patients were included. Among the fully typeable specimens (n = 197), 22 strain types were identified, with 1 type, 14d/g, accounting for 54%. The majority (93%) of the patients reported acquiring syphilis in Denmark. Among patients with concurrent HIV, 9 full strain types were identified and no difference in strain type was found by HIV status (p = 0.197). In conclusion, the majority of patients were infected in Denmark and the HIV-infected syphilis patients were diagnosed with a wide spectrum of different strain types of Treponema pallidum.