Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S–23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to ‘Mycobacterium franklinii’ DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae–Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA–DNA hybridization demonstrated that ‘M. franklinii’ DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae–M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524^T ( = ATCC BAA-2149^T) is the type strain.