Prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) infection was estimated in the ruminant populating of Uttar Pradesh (33 villages) and organized herds of Punjab (bovines) in India. In UP villages were selected randomly from the total number of villages in the country. Of the total 33 villages surveyed, 21 were in South UP and 12 in Western UP. In all 879 animals (buffaloes, cattle and goats) belonging to 700 farmers in these 33 villages were screened for paratuberculosis infection using serum samples. Prevalence of MAP infection in UP using plate ELISA (MAP Bison type antigen) was 31.9%, 37.6% and 33.3% in buffaloes (601), cattle (125) and goats (153), respectively. Prevalence in South UP was 40.3%, 42.6% and 15.0% in buffaloes (260), cattle (75) and goats (93), whereas in Western UP, it was 25.5%, 30.0% and 50.0%, in buffaloes (341), cattle (50) and goats (14), respectively. Bovine (buffaloes and cattle) and caprine samples from Ludhiana region of Punjab were from organized herds and consisted of fecal (32), milk (27), and serum (699). Prevalence of MAP infection using MAP Bison type antigen in plate ELISA was 25.0% and 15.25% in bovine (699), and caprine (177) serum samples, respectively. In milk ELISA, of 27 bovine samples 14.8% animals were positive. However, in culture 62.9% and 70.3% animals were found positive in culture of sediment and fat layers of bovines milk samples. On the basis of culture of bovine fecal samples (32), 62.5% were found positive in culture, whereas in IS900 PCR 31.25% fecal samples were positive. Culture was most sensitive than milk ELISA and PCR for the diagnosis of MAP infection in bovines and caprine population.