In this study, 25 heterologous bovine microsatellite markers have been used for the assessment of genetic diversity in Nagpuri buffalo, an important breed of Central India. For this, 48 DNA samples of unrelated individuals of Nagpuri buffalo were PCR amplified and microsatellite alleles were resolved in 6% denaturing, silver stained Urea-PAGE gel. Genotypic status of individuals at each locus was identified manually and data analysis carried out using POPGENE software. Observed number of alleles varied from 2 (ILSTS073 locus) to 8 (HEL13 & ILSTS058 loci) with a mean of 5.24 alleles per locus. Moderate level of heterozygosity (0.45) indicated sufficient genetic diversity existing in this buffalo population. PIC values for the microsatellite loci analysed, ranged from 0.10 (ILSTS019 locus) to 0.81 (ILSTS058 locus) with a mean of 0.53. No shift in the frequency distribution of alleles and a normal L-shaped curve indicated non-existence of any bottleneck in Nagpuri. The study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in buffalo breeds as well. Also various diversity indices suggest sufficient genetic variability within Nagpuri buffalo that can be utilized as initial guidelines for future breeding strategies and conservation.