Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of infectious enteritis called paratuberculosis that has a high economic impact on the worldwide livestock production. A central important question arises: Can wildlife animals serve as a reservoir for transmission of MAP to domestic ruminants? With this in mind, we devised a study to detect MAP in various Slovakian wildlife species found in the areas where paratuberculosis had been documented in domestic ruminants. The samples of parenchymatous organs (intestines, ileocecal valve and mesenteric lymphatic nodes) from 83 wildlife animals representing 13 species, inclu- ding 7 herbivorous, 5 carnivorous and 1 omnivorous species were collected during a four-year period. The clinical and pathological examinations failed to demonstrate any manifestations of paratuberculosis in any of the wildlife samples. The detection of MAP was done by widely used tests, i.e. cultivation and the PCR analysis. The bacterial cultures revealed the growth of Mycobacterium spp. colonies in 58 (70%) of all of the wild animals, but the PCR testing demonstrated paratuberculosis only in one (7.69%) of the roe deer population.