Over the past 10 years, 16S rRNA gene sequencing has contributed to the establishment of more than 45 novel species of non-tuberculous mycobacteria and to the description of emerging mycobacterial infections. Cumulative experience has indicated that this molecular tool underestimates the diversity of this group and does not distinguish between all recognized mycobacterial taxa. In order to improve the recognition of emerging rapidly growing mycobacteria (RGM), rpoB gene sequencing has been developed. Our previous studies have shown that an RGM isolate is a member of a novel species if it exhibits >3 % sequence divergence in the rpoB gene from the type strains of established species. When applied to a collection of 59 clinical RGM isolates, rpoB gene sequencing revealed nine novel isolates (15.3 %) whereas only two isolates (3.4 %) were deemed to be novel by conventional 16S rRNA gene sequence analysis. A polyphasic approach, including biochemical tests, antimicrobial susceptibility analyses, hsp65, sodA and recA gene sequence analysis, DNA G+C content determination and cell-wall fatty acid composition analysis, supported the evidence that these nine isolates represent three novel species. Whereas Mycobacterium phocaicum sp. nov. (type strain N4T = CIP 108542T = CCUG 50185T) and Mycobacterium aubagnense sp. nov. (type strain U8T = CIP 108543T = CCUG 50186T; Mycobacterium mucogenicum group) were susceptible to most antibiotics, Mycobacterium bolletii sp. nov. (type strain BD(T) = CIP 108541T = CCUG 50184T; Mycobacterium chelonae-abscessus group) was resistant to the quinolones, tetracycline, macrolides and imipenem. Only M. bolletii was resistant to clarithromycin. These data illustrate that rpoB gene sequence-based identification is a powerful tool to characterize emerging RGM and mycobacterial infections and provides valuable help in differentiating RGM at both the intra- and interspecies level, thus contributing to a faster and more efficient diagnosis and epidemiological follow-up.