BACKGROUND: Johne's disease (JD) is due to M. paratuberculosis. Nevertheless, 10% of cases could be due to M. avium subsp silvaticum. Polymerase Chain Reaction (PCR) assays developed for a rapid diagnosis of JD should also detect that mycobacteria. METHOD: Three Insertion sequences (IS) which were showed to be multi-copy elements specific for M. paratuberculosis (IS900), M. avium (IS901) and M. avium subsp silvaticum (IS902) were compared. As 4 highly conserved areas were identified, we designed 2 sets of primers for use in a nested PCR. The area spanned by the inner primers contains recognition sites for restriction enzymes which are different if the PCR product is derived from M. paratuberculosis or M. avium and M. avium subsp silvaticum. RESULTS: We tested 36 strains, including 12 M. paratuberculosis and M. avium subsp silvaticum, 8 M. avium and 16 other unrelated mycobacterial species. All of the 12 M. paratuberculosis and M. avium subsp silvaticum strains were detected and correctly differentiated by their restriction pattern. Two of the 8 M. avium strains and none of the 16 unrelated mycobacterial strains were also found to be positive. CONCLUSION: We report a single PCR assay for the detection of both M. paratuberculosis and M. avium subsp silvaticum which are then distinguished by restriction of the PCR products obtained.