The genetic subtraction technique known as representation difference analysis (RDA) PCR was applied to look for DNA and genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp DNA fragment that was used to isolate a larger genetic element found in M.avium subsp. paratuberculosis and M.avium subsp. silvaticum, both associated with enteric disease. This element, designated GS, was absent from the very closely related and relatively benign M.avium subsp. avium. It is more than 6.5 kbp in length and has a G+C content 9 % lower than other genes from this species indicating that it originates from an organism outside of the M.avium complex (MAC). A previously uncharacterized insertion sequence is associated with one end. The GS element encodes five ORFs, all of which have counterparts encoded in M.tuberculosis. and a number of bacterial species predominantly Gram negative organisms, including a number of enteric pathogens. Genes homologous to those found within GS encode functions related to the biosynthesis of LPS or extracellular polysaccharide. This element has a number of features in common with pathogenicity islands particularly its low G+C content, also its absence from a less virulent close genetic relative, M.avium, its association with an insertion sequence and the grouping of genes of related function with putative virulence linkage. No direct link to pathogenicity has been shown but GS may belong to a group of related 'genetic islands' and represents the first mobile genetic element of this class to be identified in mycobacteria.