Objective: Considering the lack of data on T. pallidum genotyping in Brazil, we aimed to study its strains and their resistance to macrolides in genital ulcers suggestive of syphilis.Methods: Men with genital ulcers suggestive of syphilis were invited to participate. Samples were collected with a dry cotton swab and immersed in a 0.9% NaCl solution. Detection was done by PCR amplification of 260 bp of the tpp47 gene. The PCR product was analyzed by electrophoresis in a 2% agarose gel containing 0.05% ethidium bromide. Positive PCR samples were analyzed by MLST (sequencing of chromosomal loci TP0136, TP0548, and TP0705). The A2058G and A2059G mutations in the 23S rRNA gene were evaluated by nested PCR. DNA sequencing was analyzed using Bioedit software (Tom Hall, USA). Genotyping was performed using the PubMLST online platform (Grillová scheme). Results: All subjects were residents of Porto Alegre and aged between 19 and 66 years. Of the 43 samples, 32 were positive for PCR for T. pallidum. Thirty strains were available for genotyping and belonged to the SS14-like (73.3%) or Nicholslike (20%) Clonal Complex. Three complete MLST profiles were identified (1.3.1; 9.7.3 and 28.7.3), and a new allele was identified in one sample (approved by pubMLST curators as TP0705-22). Only one sample did not present the 2058 mutation in the 23S rRNA gene.Conclusion: Our study identified genetic diversity in T. pallidum DNA using MLST with allelic variants for TP0136, TP0548, and TP0705, including a new allele. A single sample was characterized as genotypically susceptible to macrolides. All other samples (more than 95%) presented the A2058G mutation in the 23S rRNA gene, which causes resistance to macrolides. Improving understanding of the local epidemiology of T. pallidum with representative samples that allow cofactor analysis is crucial for prevention and care.