The objectives of the present study were to characterize the rarely studied gross and histological lesions of paratuberculosis in water buffaloes and to study the efficacy of the different diagnostic methods. Intestinal tissues (n=405) including jejunum, ileum, ileocecal junction and associated mesenteric lymph nodes (MLN) collected from 1000 buffaloes screened at slaughterhouse and 5 buffaloes necropsied at the postmortem room of the institute were examined histologically for paratuberculosis lesions. Selected tissues (n=50) were subjected to bacterial culture and IS900 PCR. On histological evaluation of sections, granulomatous inflammatory changes were observed in 20 animals that had shown gross lesions of intestinal thickening and mucosal corrugations, and enlarged and edematous mesenteric lymph nodes. Histological lesions were classified into 3 grades (Grade-1, 2 and 3) on the basis of type and severity of cellular infiltrates and presence of acid-fast bacilli (AFB) or acid-fast granular debris. Grade-1 lesions observed in 8 animals were characterized by scattered infiltration of epithelioid macrophages amongst large number of lymphocytes with macrophages containing acid-fast granular debris in the intestine and lymph nodes. Grade-2 lesions (n=8) consisted of microgranulomas in the intestine and large granulomas in the MLN with AFB or acid-fast granular debris. Grade-3 lesions (n=4) were characterized by the presence of multiple granulomas and multinucleated giant cells and definite AFB in the intestine and MLN. Positive bacterial culture and IS900 PCR was observed in 6 and 14 animals, respectively. The partial sequence analysis of PCR products of bubaline isolate of Mycobacterium avium subsp. paratuberculosis (Accession No: AY 660657) showed more than 98% of homology with the published sequence of IS900 gene (Accession No: X16293). From the results of the present study, it was concluded that the histological lesions observed in water buffaloes could be paucibacillary type and tissue PCR was found to have more sensitivity than bacterial culture and smear examination.