A DNA sequence differing between sheep and cattle types of Mycobacterium avium subsp. paratuberculosis was identified and used to develop a PCR test. The test unequivocally distinguished all sheep types from cattle types and was negative for a wide range of other strains from the Mycobacterium avium-Mycobacterium intracellulare complex. The test will be useful for epidemiological purposes, particularly in hosts such as deer that can be easily infected with either type.