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Background: The province of Manitoba, Canada, with a population of approximately 1.3 million, has been experiencing increased incidence of syphilis cases since 2015. In this study, we examined the detection of Treponema pallidum DNA in 354 clinical samples from 2012 to 2016, and determined molecular types and mutations conferring resistance to azithromycin in the polymerase chain reaction (PCR)-positive samples.Methods: T. pallidum DNA detection was done by PCR amplification of tpp47, bmp, and polA genes. Syphilis serology results were reviewed for the PCR-positive cases. Molecular typing of syphilis strains was done by analysis of the T, pallidum arp, tpr, and tp0548 gene targets as well as partial sequencing of the 23S rRNA gene for azithromycin resistance.Results: Of the 354 samples tested, 74 individual cases were PCR positive. A result from the treponemal antibody chemiluminescent microparticle immunoassay test was positive in 72 of these cases and that from the Venereal Disease Research Laboratory testing was positive in 66. Mutations conferring resistance to azithromycin were found in all 74 PCR-positive samples. Molecular typing was completed on 57 PCR-positive samples, and 12 molecular types were identified with 14d/g found in 63.2%. Increased strain diversity was observed with 8 molecular types detected in 2016, whereas only 2 to 3 types were found in 2012 to 2014. A patient with 2 episodes of infection 9 months apart caused by different molecular strain types was also identified.Conclusions: The finding of an increase in genetic diversity in the strains in this study and an increase in macrolide resistance compared with previous Canadian reports highlighted the need for continued surveillance including strain characterization. [ABSTRACT FROM AUTHOR]