Johne's disease (JD), caused by Mycobacterium paratuberculosis, is a chronic enteritis and lymphadenitis affecting ruminants and camelids. JD is found in most countries in the world and is present in Australia. During surveillance work in Western Australia and test and cull procedures in Victoria, 4 isolates that were not Mycobactin dependent were found to be IS900 PCR positive. Restriction digest of the amplified products confirmed that the product was not consistent with the sequence of M. paratuberculosis. The isolates were further investigated using IS900 and 16S rRNA sequencing, and by restriction fragment length polymorphism (RFLP) using IS900 as probe. Sequence differences were detected between the two WA isolates but the two Victorian isolates were identical using 16S and IS900 sequencing. Using 16S sequencing, all isolates most closely resembled an unclassified IWGMT strain. Sequence differences were detected between the three strains within the amplified product of IS900 and homologies of between 73 and 83% were present within the IS900 target. In each case, sequencing confirmed that the selected restriction sites in these isolates were not consistent with M. paratuberculosis. Between 3 and 5 copies of the IS900-like sequences was confirmed by RFLP. Although it is accepted that there is homology at the 3' end of IS900 between M. paratuberculosis and other M. avium spp, the 5' end of this IS has previously been considered specific for M. paratuberculosis. The isolation and characterisation of these isolates has confirmed considerable homology with M. paratuberculosis in this region. The finding of these organisms suggests that IS900 PCR may be less than 100% specific and it is suggested that where such technology is used for identification of M. paratuberculosis, a restriction digest be applied to confirm the internal sequence of the amplified product.