Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.