Background: In Canada, the recent resurgence of infectious syphilis and rising rates of congenital syphilis have renewed interest in direct detection methods for the laboratory diagnosis of syphilis. The Canadian Public Health Laboratory Network (CPHLN) has previously published a series of guidelines for the diagnosis of syphilis in Canada, including the use of direct tests. In the decade since those guidelines were published, laboratory practice has changed. Methods: This systematized review combined a literature search (MEDLINE) of methods for direct detection of Treponema pallidum with an informal survey of current testing practices in Canadian public health laboratories. Results: Direct testing methods have favourable performance characteristics for detection of early syphilis and congenital cases. Although no government licensed commercial nucleic acid amplification test (NAAT) for syphilis is available in Canada, laboratory-developed tests have been implemented in multiple Canadian jurisdictions. Clinical specimens with the highest yield of positive NAAT results for syphilis include genital ulcers, skin lesions, and oral swabs from primary and secondary syphilis patients. For investigation of congenital syphilis, nasopharyngeal, placenta, umbilical cord, blood, and skin lesions are specimens of choice for direct detection of T. pallidum by NAAT. Conclusions: This update on the status of direct testing highlights the importance of NAAT for the detection of T. pallidum, the reduced role of microscopy-based methods, and the emergence of DNA and genome sequencing as tools for phylogenetic analysis and molecular epidemiology.