Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.