To classify a specific Mycobacterium among various mycobacteria utilizing sequencing of rpoB gene. To classify mycobacteria not identified by DNA-DNA hybridization (DDH) using sequencing of rpoB and 16S rRNA gene.Classification of 106 Mycobacteria strains, one Nocardia strain, one Rhodococcus strain, four Gordona strains was made by using partial sequencing of rpoB and 16S rRNA (RIDOM). Thereafter, 38 mycobacteria clinical strains not identified by DDH were classified utilizing the DNA sequencing data.Pairs of M. kansasii and M. gastri, M. abscessus and M. chelonae, M.fortuitum (ATCC49404) and M. polcinum, M. peregrinum and M. septicum, M. farucinogense and M. senegalense and M. fortuitum (ATCC49403), Rhodococcus, Nocardia and Gordona strains were classified using sequencing of rpoB gene. Even though sequencing of rpoB and 16S rRNA gene was utilized, it was impossible to classify M. tuberculosis complex, M. avium family, M. marinum and M. ulcerans, and M. fortitum subsp. fortuitum and M. fortuitum subsp. acetamidolyticus. The 38 mycobacteria clinical strains not identified by DDH were successfully classified using sequencing of both rpoB and 16S rRNA. These sequencing analyses showed that M. heckeshornense, M. branderi, M. intermedium, M. shimoidei, M. wolinskyi, M. malmoense and M. lentiflavum could be identified. Thirty six clinical isolates (94.7%) and 32 clinical isolates (84.2%) were identified by rpoB sequencing and 16S rRNA sequencing (RIDOM), respectively.The classification ratio of mycobacteria including Nocardia, Rhodococcus and Gordona is 69.6% for sequencing of 16S rRNA and 89.3% for sequencing of rpoB gene. Sequencing of rpoB is useful for classification of mycobacteria due to its genetic diversity, but has some limitation in its application. In order to classify mycobacteria more accurately, it is important to combine sequencing of rpoB and 16S rRNA and biochemical/biological tests.