Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD or paratuberculosis) in animals and has also been implicated in Crohn's disease of humans. It has been shown that MAP is endemic in animal population of India. Understanding of heterogeneity among MAP strains is important both for diagnosis and design control measures. Genotyping and epidemiological investigations revealed that MAP 'Bison type' was the predominant strain infecting domestic ruminant population in India. MAP 'Bison type' has also been reported from USA. A number of comparative genomics studies have been conducted to understand 'Cattle type' and 'Sheep type' strains. However, present study was the first attempt to characterize MAP 'Bison type' S5 using different markers including IS900, ISMAP02, IS1311, LSPs and SSRs. Study showed that MAP S5 is similar to MAP K10 in terms of number of IS900, IS1311 and ISMAP02 elements. There was high sequence similarity for IS900 and ISMAP02 between MAP K10 and MAP S5. However, this study also reported genetic differences between two strains. In some IS1311 loci, TG gap at 64th and 65th position was observed in MAP S5. Further sequencing of few more MAP isolates confirmed that this gap was specific to indigenous MAP 'Bison type' and can be further used as molecular signature. ISMAP02 locus 1 was observed at polymorphic position in MAP S5 compared to MAP K10. MAP 'Bison type' S5 also showed polymorphic profile for LSP(P)4. Polymorphism was also observed in SSRs. This pilot study may form the basis for future epidemiological investigations.