Abstract: The present invention provides oligonucleotides useful as amplification primers and assay probes for species-specific detection and identification of Mycobacterium kansasii. The oligonucleotides of the invention are derived from the p6123 sequence previously reported by Yang, et al. (1993) and amplify a target sequence of both atypical and typical M. kansasii strains, allowing detection and identification of a broad range of M. kansasii strains. The oligonucleotides may be used after culture as a means for confirming the identity of the cultured organism or they may be used prior to culture or in place of culture for detection and identification of mycobacterial nucleic acids using a variety of amplification methods.