Eradication and control programs of Johne's disease (JD) in Australia assume that cattle are not susceptible to infection with the sheep strain of M. avium subsp. paratuberculosis and can safely graze on pasture with or after the removal of sheep affected with ovine Johne's disease. Therefore, ongoing strain identification of JD cases is essential to substantiate this assumption. Currently diagnosis of JD is confirmed by Polymerase chain reaction (PCR) on IS900, an insertion sequence (IS) unique to M. avium subsp. paratuberculosis and strain identification is achieved by restriction fragment length polymorphism (RFLP) analysis, an expensive and time consuming process. Recently, another insertion sequence, IS1311, was found in M. avium subsp. paratuberculosis and M. avium. Characterisation of IS1311 in sheep and cattle strains of M. avium subsp. paratuberculosis revealed several point mutations compared to the M. avium sequence and these can be used to discriminate between the species. In addition, a polymorphic IS1311 locus in the cattle strain of M. avium subsp. paratuberculosis can be used to differentiate it from the sheep strain. A rapid and sensitive PCR- test based on IS1311 has been developed and validated on an extensive range of M. avium subsp. paratuberculosis and M. avium isolates from a variety of sources including primary radiometric cultures, purified DNA and crude DNA from cultured organisms. With this test we confirmed the presence of M. avium subsp. paratuberculosis or M. avium and achieved a 100% correlation with RFLP or species of origin.