<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>(<it>M. avium</it>) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.</p> <p>Results</p> <p>A comparative genomic approach was used to identify large sequence polymorphisms among <it>M. avium </it>subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate <it>M. avium </it>subsp. <it>paratuberculosis </it>(MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. <it>M. avium </it>subsp. <it>silvaticum </it>isolates were observed to have a hybridization profile very similar to yet distinguishable from <it>M. avium </it>subsp. <it>avium</it>. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.</p> <p>Conclusion</p> <p>Genome diversity in <it>M. avium </it>subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of <it>M. avium </it>were distinguishable by the presence or absence of specific polymorphisms.</p