The PCR systems directed against the IS900 gene have proved very useful for the identification of Mycobacterium paratuberculosis. However, when IS900-like genes were found in other unrelated Mycobacterium species, it was revealed that these PCR systems are not completely specific for M. paratuberculosis. It is therefore important to investigate alternative PCR systems for confirmation of positive IS900 PCR tests. A duplex PCR system (Coetsier et al 2000) targeting the p34 gene and the f57 gene was applied to 91 strains of M. paratuberculosis and other mycobacteria from different animals and countries. Two fragments of expected sizes were detected from the 60 tested M. paratuberculosis strains and one fragment, from the amplified p34 gene, was found in the 16 M. avium strains. Strain 2333, with an IS900-like gene, gave no fragment in the p34/f57 PCR. One strain each of M. tuberculosis and M. bovis both resulted in a fragment from the p34 gene, slightly shorter than the corresponding fragments from M. paratuberculosis and M. avium. Fragments of the amplified p34 gene were found in 8 of the other mycobacterial strains tested. The p34/f57 PCR seems suitable for confirmation of samples tested positive for M. paratuberculosis in IS900 PCR-systems. A novel fingerprinting PCR system representing a reproducible randomly amplified polymorphic DNA (RAPD) was developed and applied to strains of M. paratuberculosis and other mycobacteria. The PCR is based on primers targeting the enterobacterial repetitive intergenic consensus (ERIC) elements and IS900. Sixty M. paratuberculosis strains, 16 M. avium and 15 other mycobacteria, including the IS900 positive strain 2333, were tested. Reproducible fingerprints were obtained for all mycobacteria analysed. The M. paratuberculosis strains all exhibited the same banding pattern, clearly different from the fingerprints of strain 2333 and other mycobacteria. This PCR-system offers an alternative to IS900 PCR for identification of M. paratuberculosis.