Mycobacterial interspersed repetitive units (MIRUs) are short repetitive DNA elements found as tandem repeats in intergenic regions of mycobacterial genomes. MIRUs display variations in tandem repeat copy numbers and exhibit minor sequence variations between repeat units. Although MIRUs may be useful for fingerprinting, their function remains largely unknown, however it has been proposed that they may play a role in genomic structure and rearrangements, differential translation of genes, mRNA stability and transcriptional termination. Using a 53bp MIRU consensus sequence from Mtb BLAST searched onto Mycobacterium avium subspecies avium (Maa) 104 genome we have identified 25 MIRU loci each containing 1-5 tandem MIRU copies. Using this data we BLAST searched a Maa MIRU consensus onto the Mycobacterium avium subspecies paratuberculosis (Map) genome and identified a total of 16 MIRU loci, 9 of which contained single MIRU with 100% homology to Maa. Seven MIRU were found to display variations in arrangement and copy number compared to the corresponding loci in Maa. Based on these polymorphic loci, we have developed a PCR-based typing method for Maa, Mycobacterium avium subspecies silvaticum (Mas) and Map strains. When MIRU PCR was applied to a panel of Maa, Mas and Map this showed 100% differentiation of species and 4 different profiles amongst Map strains. Results of MIRU PCR on Map vaccine strains also demonstrate a unique MIRU profile. This work demonstrates a new heterogeneity of Map strains and provides a new method for the molecular identification of Maa, Mas and Map.