Background & objectives: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), ′Bison type′ is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn′s disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between ′Indian Bison type′ and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. Methods: A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. Results: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to ′Bison type′. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as ′Bison type′, ′Cattle type′ and ′Sheep type′, respectively. IS1311 L2 PCR-REA method showed different restriction profiles of ′Bison type′ genotype as compared to non-Indian DNA samples. Interpretation & conclusions: IS1311 L2 PCR-REA method successfully discriminated ′Indian Bison type′ from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.