An acid-fast, rapidly growing, psychrotolerant short rod was isolated from pond water near a uranium mine. Phylogenetic analysis of the 16S rRNA gene sequence grouped this strain with the rapidly growing mycobacteria. The 16S rRNA gene sequence of isolate WA101T showed highest similarity to that of Mycobacterium sphagni DSM 44076T; however, DNA-DNA relatedness between the two strains was less than 30 %. Chemotaxonomic analyses, which included fatty acid and mycolic acid patterns, confirmed the classification of strain WA101T in the genus Mycobacterium. Physiological data, including antibiotic resistance, NaCl tolerance, carbon sources, temperature growth range and enzyme activities, were also determined. Based on the genotypic and phenotypic results it is proposed that isolate WA101T represents a novel Mycobacterium species. The name Mycobacterium psychrotolerans sp. nov. is proposed, with type strain WA101T (= DSM 44697T = LMG 21953T).