All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.