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Several intermediate-growing, photochromogenic bacteria were isolated from sphagnum peat bogs in northern Minnesota, USA. Acid-fast staining and 16S rRNA gene sequence analysis placed these environmental isolates in the genus Mycobacterium , and colony morphologies and PCR restriction analysis patterns of the isolates were similar. Partial sequences of hsp65 and dnaJ1 from these isolates showed that Mycobacterium arupense ATCC BAA-1242T was the closest mycobacterial relative, and common biochemical characteristics and antibiotic susceptibilities existed between the isolates and M. arupense ATCC BAA-1242T. However, compared to nonchromogenic M. arupense ATCC BAA-1242T, the environmental isolates were photochromogenic, had a different mycolic acid profile and had reduced cell-surface hydrophobicity in liquid culture. The data reported here support the conclusion that the isolates are representatives of a novel mycobacterial species, for which the name Mycobacterium minnesotense sp. nov. is proposed. The type strain is DL49T ( = DSM 45633T = JCM 17932T = NCCB 100399T).