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Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, a disease with considerable economic impact on dairy buffalo herds. The present study was undertaken to assess the prevalence of MAP infection in buffaloes and its excretory pattern through buffalo milk. A total of 74 milk samples were collected from apparently healthy buffaloes of organized and unorganized sectors located in Tamil Nadu and subjected to Ziehl-Neelsen staining and Polymerase chain reaction (IS900 and F57 genes). Out of 74 samples, 3 (4.1%), 21 (28.4%) and 14 (18.9%) samples shed MAP organism by Ziehl-Neelsen staining, IS900 PCR and F57 PCR respectively. Besides age of the animal, stage of lactation and herd management were associated with excretion of MAP in milk. These results showed the high prevalence of MAP infection in buffaloes and warrants further studies and necessary actions to delineate the MAP infection in buffalo population.