In ruminant, Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic persistent infection in small and large intestine, resulting in chronic persistent diarrhea. It is also known as paratuberculosis (JD). It is increasingly being recognized as a problem that adversely affects animal health leading to significant economic losses to livestock industry. Recent emerging evidences have established a link between MAP and inflammatory bowel disease (IBD), Crohn’s disease (CD) suggesting its Zoonotic importance hence the present study was designed to find out percent incidence of MAP infection in buffaloes from Malwa region of Madhya Pradeh by using faecal microscopy. The study was conducted in buffaloes (females and males), belonging to different region of Malwa tract viz. Depalpur, Dewas, Mhow, Dhar (Sundrel), Indore and Maheshwar ranging between 1 to 10 years of age, slaughtered at Cantonment Board slaughter house, Mhow and Nagar Nigam Indore. Based on the presence of pink colored short rod (0.5-1.5 micron) shaped MAP bacteria by feacal smear examination revealed 79.33% (119/150) incidence of MAP in buffaloes of Malwa region.