The lack of methods for differentiating M. avium ssp. paratuberculosis isolates has hampered epidemiological studies investigating the spreading of the disease. To contribute to solving this problem we have evaluated a set of 60 decamer primers with a GC-content above 50% alone and in combination in a RAPD-PCR. We succeeded in identifying 10 primers which reproducibly resulted in PCR-products of 200 to 600 bp. Subsequently, DNA was prepared from 12 clinical M. avium ssp. paratuberculosis isolates. Based on these results it should be possible to investigate the origin of isolates in newly infected herds.