Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).