The genus Mycobacterium contains the pathogenic species tuberculosis and leprae, and additionally at least 148 sometimes opportunistic pathogenic species that occupy environmental niches. The genus Mycobacterium is in the order Actinomycetes along with other acid-fast genera with mycolic acids in their cell walls, such as Corynebacterium, Nocardia, and Rhodococcus. The DNA-dependent RNA polymerase subunit b gene rpoB, is relatively conserved among these bacteria. Sequence polymorphism in a 342bp fragment of rpoB is sufficient to differentiate among most species of Mycobacterium. However, construction of a robust and testable phylogeny or population structure based on this polymorphism, or polymorphism in other genomic regions, has remained elusive. The correctness of the MST-regions model of genus Mycobacterium population structure was validated by statistically confirmed linkage disequilibrium of certain restriction site alleles. For certain alleles, SGM in MST-region 1 resembled the RGM of MST-region 1 more than the SGM of MST-region 3. The model provided a framework consistent with published genotypic and phenotypic observations, including expectations from comparative genomics and ribosomal RNA (rRNA) gene properties’ distribution among species, and reported cladistic groupings of species.