The newly formulated Mycobacterium kansasii AccuProbe was evaluated, and the results obtained with the new version were compared to the results obtained with the old version of this test by using 116 M. kansasii strains, 1 Mycobacterium gastri strain, and 19 strains of several mycobacterial species. The sensitivity of this new formulation was 97.4% and the specificity was 100%. Still, three M. kansasii strains were missed by this probe. To evaluate the variability within the species, genetic analyses of the hsp65 gene, the spacer sequence between the 16S and 23S rRNA genes, and the 16S rRNA gene of several M. kansasii AccuProbe-positive strains as well as all AccuProbe-negative strains were performed. Genetic analyses of the one M. gastri strain from the comparative assay and of two further M. gastri strains were included because of the identity of the 16S rRNA gene in M. gastri to that in M. kansasii. The data confirmed the genetic heterogeneity of M. kansasii. Furthermore, a subspecies with an unpublished hsp65 restriction pattern and spacer sequence was described. The genetic data indicate that all M. kansasii strains missed by the AccuProbe test belong to one subspecies, the newly described subspecies VI, as determined by the hsp65 restriction pattern and the spacer sequence. Since the M. kansasii strains that are missed are rare and all M. gastri strains are correctly negative, the new formulated AccuProbe provides a useful tool for the identification of M. kansasii.