Conventional tests for the identification of mycobacteria may frequently result in erroneous identification and underestimate the diversity within the genus Mycobacterium. However, this problem can be overcome by molecular approach like as 16S rRNA gene (rDNA) or RNA polymerase gene (rpoB) sequence analysis. In this study, a molecular approach analyzing partial sequence of 16S rDNA and rpoB gene was applied to mycobacteria other than M. tuberculosis (MOTT) isolates that had not been definitely identified by conventional physical and biochemical tests. Among the eighteen isolates included in this study, twelve isolates could be identified to the species level and six were identified to the complex level. Compared with the results by 16S rDNA analysis, the rpoB analysis could differentiate some of the strains into the subspecies level.