Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.