Serological test for primary syphilis could be negative the first 5-15 days. The aim of this study was to evaluate the benefit of including dark field microscopy (DFM) in the diagnosis algorythm for primary syphilis. Patients attended to a sexual transmission diseases clinic of Madrid, from 2015 to 2019, for a genital ulcer with clinical suspicion of primary syphilis. They were tested for DMF and serological test (EIA/TPPA/RPR). Over the total amount of samples (806), 53.2% (429) were positive for DFM. Thus, the 48% of the 429 patients had negative serological test (EIA/RPR) of which the 77.6% were positive at TPPA. DFM allows primary syphilis early diagnosis, even without serological test. If no direct detection methods are available, for patients without history of syphilis, TPPA could help to diagnose primary syphilis.