MLVA methods for strain discrimination among Mycobacterium tuberculosum strains are disclosed. Nine VNTR loci have been identified from genomic sequences of Mycobacterium tuberculosum strains and primer pairs suitable for amplifying the VNTR by PCR are disclosed. Polymorphisms at these loci were used to resolve genotypes into distinct groups. This sub-typing scheme is useful for the epidemiological study of Mycobacterium tuberculosum and may be applied to the local detection of the pathological causative agent of tuberculosum.