Abstract: The present invention relates to a method for detecting and identifying mycobacterial species which comprises steps of amplifying 342 bp of rpoB gene fragments from clinically isolated mycobacterial using mycobacterial rpoB-specific PCR primers; sequencing 306 bp regions of the amplified 342 bp of rpoB gene fragments except the primer regions; and, inferring a phylogenetic tree with reference species. In accordance with the present invention, it was found that rpoB sequences from 44 mycobacterial species provide a basis for systematic phylogenetic relationship which can be used to identify clinically isolated mycobacteria that are pathogenic or potentially so. Accordingly, the amplification of rpoB DNA followed by automated sequencing and the analysis of phylogenetic relationships with the reference species can be used efficiently to detect and identify clinical isolates of mycobacteria which have not been identified by the conventional methods.