Uttar Pradesh is the fourth largest, most populous and leading milk and meat producing state in India. Despite the huge livestock population, information on the status of paratuberculosis homogeneity and heterogeneity of Mycobacterium avium subspecies paratuberculosis (MAP) isolates of eastern Uttar Pradesh is non-existent. Present study was aimed to estimate the presence of MAP in large ruminants (Cattle and Buffaloes) of eastern Uttar Pradesh. A total 108 fecal samples were collected from farmer's herds of large ruminants (cattle and buffaloes) from different geographical regions (Chandauli, Mughalsarai, Gazipur, and Naugarh) of eastern Uttar Pradesh and screened for the presence of MAP infection using microscopic examination, direct IS900 PCR and culture on Herrold egg yolk (HEY) medium. The isolates recovered on HEY medium were subjected to molecular identification and genotyping using IS900 PCR and IS1311 PCR-REA method, respectively. Of the 108 fecal samples, 25 (23.14%) and 11 (10.18%) samples were positive for the presence of acid-fast bacilli and growth on HEY medium, respectively. Species-wise, 17.5, 7.5% and 26.5, 11.7% fecal samples from cattle and buffaloes were found positive for the presence of acid-fast bacilli and growth on HEY medium, respectively. Isolates recovered on HEY medium with mycobactin J were positive for IS900 sequence and genotyped as Bison Type using IS1311 PCR-REA method. Present study is the first report on the presence of MAP infection and ‘Bison Type' genotype of MAP in eastern Uttar Pradesh. These findings will be useful for the intervention of effective control measures in order to reduce the prevalence of MAP infection in domestic livestock species and prevent its spread to the human population in the regions