The whole-genome sequencing of a bovine isolate of Mycobacterium avium subspecies paratuberculosis (MAP) and subsequent annotation has identified all of the putative open reading frames (ORFs) encoded in the genome. However, it remains unclear if the genomic content of this single isolate is representative of other bovine isolates as well as isolates from other species. To investigate this problem, we have designed and built a DNA microarray consisting of PCR products representing over 95% of the ORFs identified in the MAP K10 genome sequence. Genomic DNA from MAP isolates was fluorescently labeled, mixed with alternately labeled MAP K10 DNA, and competitively hybridized on the MAP microarray. ORFs were classified as present, divergent, or intermediate based on the relative fluorescent intensities compared to MAP K10 DNA. MAP isolates cultured from cattle, bison, sheep, goat, mink, and human sources were hybridized to the MAP microarray. Of the isolates examined thus far, only sheep isolates contained any ORFs classified as divergent. Three putative deletions were observed in the genomes of two MAP isolates obtained from sheep. The first deleted region includes the ORFs MAP1433c through MAP1437c which encode dehydrogenase and oxygenase enzymes. The second deleted region was found to contain the ORFs MAP1487c through MAP1490 which encode dehydrogenase enzymes. The third and largest deleted region was observed from MAP1728c through MAP1744 and includes ORFs encoding an esterase, membrane proteins, regulatory proteins, a PPE family protein, and several hypothetical proteins with no predicted function. Additionally, differences in hybridization were detected for the microarray targets representing the insertion sequence ISMAP04, suggesting that there may be an absence or change in the copy number of this element relative to the MAP K10 genome. These studies have revealed that with the exception of ovine MAP isolates, the genome content of MAP K10 is conserved across bovine MAP isolates as well as several other host species.