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Mycobacterium subspecies paratuberclosis is an important zoonotic pathogen responsible for Johne's and Crohn's diseases in animals and humans population, respectively. Foods with animal origins may act as reservoirs of M. avium subsp. paratuberclosis. The present survey was done to assess the molecular distribution of M. avium subspecies paratuberculosis in the milk of cow, sheep, goat, donkey, buffalo, and camel species. In this study, a total of 300 raw milk samples were collected from Chaharmahal Va Bakhtiary and Isfahan province, Iran. Samples were subjected to DNA extraction. Extracted DNA was assessed for the M. avium subspecies paratuberculosis genome using the Polymerase Chain Reaction. Thirty out of 300 (10%) raw milk samples were positive with M. avium subsp. paratuberculosis. Raw buffalo milk samples harbored the highest contamination rate of M. avium subsp. paratuberculosis (17.50%), while raw donkey milk samples harbored the lowest (5%). Total prevalence of M. avium subspecies paratuberculosis amongst the raw milk samples collected from Isfahan and Chaharmahal Va Bakhtiary province were 66.66% and 33.33%, respectively. Statistically significant differences were obtained between types of samples and bacterial distribution (P <0.05) and also geographic area of sampling and bacterial distribution (P <0.05). Role of raw milk, particularly raw buffalo milk samples as reservoir of M. avium subsp. paratuberculosis have been determined. Using pasteurized and sterilized milk will decrease the risk of M. avium subsp. paratuberculosis.