Syphilis, caused by Treponema pallidum, is a sexually transmitted disease which was epidemic in Europe between the 15th and 20th century. From 2000 onwards it is worldwide mostly encountered among men who have sex with men but also among women in resource poor setting. Syphilis can easily be treated with penicillin but can become chronic, if left untreated. The disease develops in several stages with ulcerative lesions in the primary stage, and systemic spread to organs via the blood in later stages. Taking swab samples from these ulcerative lesions gives the best options for detection by PCR of T. palillidum. Alternatively blood samples can be analyzed in later stages. If tested positive in screening PCR assays, T. pallidum can also be typed by molecular methods, enabling molecular epidemiology. Different protocols are discussed in this chapter, that include all steps from sample collection, nucleic acid extraction, PCR, and detection. The combination of all steps together determine the final sensitivity and specificity of T. pallidum PCR.