Background: The analysis of genetic diversity of Treponema pallidum subspecies pallidum (TP) strains is a pivotal tool to investigate syphilis molecular epidemiology, transmission dynamics and mutations possibly associated with antibiotic resistance. Among currently used genotyping methods is the Enhanced CDC typing scheme (ECDCT). In parallel, assessing the presence and prevalence of mutations possibly associated with resistance to second-line antibiotics can help decide on viable treatment options. Here, we report on the distribution of TP strain types in samples from Peruvian active syphilis patients and on genetic polymorphisms that could be associated with tetracycline resistance. Methods: We analyzed DNA extracted from 87 lesion swab samples. The presence of TP DNA was assessed by amplification of three TP-specific genes, namely tp0548, tp0574, and polA (tp0105) genes. Typing of positives samples was performed by detecting the number of repeats in the arp gene, analysis of the tprE/G/J RFLP pattern, and sequence of a fragment of the tp0548 gene. The possibility of a strain genetically resistant to tetracycline was assessed among 22 samples by sequencing of a specific region of the 16S rRNA gene to search for mutations at gene positions 965 and 1058. Positivity was defined as a sample with any of the point mutations. Results: Of the 51 samples with TP-DNA, 15 (29.4%) samples yielded complete genotypes, while 16 (31.3%) could be partially genotyped, and 20 (39.3%) could not be genotyped. Among the fully-typed samples, strain type 14d/g was the most prevalent (4/15; 27%), followed by strain types 14d/d (2/15; 13%), and 12d/g (2/15; 13%). None of the 22 TPpositive samples exhibited the mutations in the 16S rRNA gene. Conclusions: The ECDCT methodology allows us to improve the still limited knowledge of molecular types of TP in Peru, despite the small number of fully typable samples. Mutations associated with decreased susceptibility to tetracycline were not detected, supporting that resistance to this antibiotic has not started to emerge in this pathogen. Hence, tetracycline continues to be useful for syphilis treatment and can be considered as preventive treatment for pre- or post-exposure prophylaxis given the recent results in doxyPrEP and doxyPEP studies. [ABSTRACT FROM AUTHOR]