Nested polymerase chain reaction (PCR) using the Mycobacterium avium subspecies paratuberculosis (Map)–specific region, locus 251, was used as a screening tool for the detection of Map DNA in fecal samples from northern Canadian bison herds. Further characterization of positive samples (26/835) was performed because Map DNA was found without signs of disease. Strain typing, using PCR-Restriction endonucleas assay (REA), was limited to two samples but revealed that the samples corresponded to a cattle-related strain and a sheep-related strain. Sequencing of part of the IS1311 region from the two samples revealed a unique three base-pair region, which is only found within the northern Canadian bison isolates.