Bacteriologic culture is considered the "gold standard" method for diagnosing paratuberculosis infection. However, bacteriologic culture of MAs paratuberculosis is time consuming and laborious and the success of bacteriologic culture varies with the species of animals tested. Improved diagnostic tests are needed that can be used in domestic and wild ruminants. PCR has been used for the detection of MAs paratuberculosis DNA in faeces and tissues. However, elaborate specimen preparation protocols are used and may result in reduced sensitivity of these assays. We applied the PCR technique to detection of paratuberculosis in twenty-five free-ranging bison. We report the performance of preassembled PCR mixtures to detect MAs paratuberculosis DNA in frozen ileum, jejunum, and mesenteric lymph nodes from bison. Specific oligonucleotide primers used in the PCR assay were derived from the 16S rRNA (MAs) sequence and an insertion element IS900 (MAs paratuberculosis). Crude genomic DNA samples from tissues were prepared using a simple boiling technique and the samples were tested by PCR. An animal was considered positive if MAs paratuberculosis DNA was detected by PCR amplification in at least two frozen tissues from an animal using the IS900 primer set or by detection of DNA in a single tissue from an animal with both the 16S rRNA and IS900 primer sets. Using these criteria, 15 of 25 (60%) bison tested were positive for MAs paratuberculosis DNA. The data indicate that these free-ranging bison had been exposed to MAs. paratuberculosis.