A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA (RAPD). A primer OPE-20 (5'-AAC-GGT-GAC-C-3') was identified to be the most suitable primer when tested with four ATCC reference strains of M. paratuberculosis and eight well characterized field strains each of M. paratuberculosis and M. avium. Primer OPE-20 was further tested for its ability to identify and subtype 200 field isolates of M. paratuberculosis. The fingerprint patterns of M. paratuberculosis (n=212) consisted of five unique common fragments (620, 450, 310, 230, 180bp) and nine variable fragments resulting in six distinct genotypes. The DNA fingerprints of M. avium (n=8) consisted of a single common fragment of 620bp, and 15 variable fragments resulting in six different genotypes. The cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a different RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible and reliable technique for identification and sub-typing of M. paratuberculosis.