Syphilis is a systemic and sexually transmitted infection caused by Treponema pallidum ssp. pallidum. This spirochete causes different clinical and subclinical stages depending on the duration of infection and immune status of the host. Several tests have been developed for diagnosis, and are classified into direct and indirect methods. The first one includes dark field microscopy, direct fluorescent antibody test in fluids or tissue, and molecular biology techniques. In the indirect method (serologic), the routine tests are used, and are divided in two categories: non-treponemal and treponemal ones. The objective of this work was to identify T. pallidum ssp. pallidum in paraffin-embedded skin biopsies positive by immunohistochemistry, using conventional polymerase chain reaction (PCR) and quantitative real time PCR (qPCR). We included a sample of 17 paraffin-embedded biopsies. DNA was extracted and processed by conventional PCR and real-time PCR with a TaqMan® probe to identify the polA gene. Using PCR, 11 tested positive (64.7%) and 6 (35.3%) were negative. With qPCR and TaqMan® probe, 100% of samples tested positive. The minimum number of spirochetes detected in each sample was 2. With this work, we can conclude that qPCR is a fast and very accurate method for diagnosis of syphilis in tissue specimens.