Diara buffaloes are dual purpose lesser known population distributed around south and north Gangetic plains of Bihar. These buffaloes are adapted to environmental conditions of Bihar, India and its genetic constitution getting diluted due to use of semen of other breeds. The phenotypic and genetic characterization studies assist to policy makers to take decisions for conservation and rational implementation of breeding programme. A total of 200 animals from all surveyed villages were included to record information on various management practices, physical characteristics and production and reproduction performances using structured questionnaire. The molecular characterization using FAO recommended 10 microsatellite markers were carried out on 50 unrelated individuals of buffaloes. Different measures of genetic diversity were estimated using various softwares. The average number observed of alleles (Na), Effective number of alleles (Ne), Shannon Information Index (I), Observed heterozygosity (Ho), Expected heterozygosity (HE), Average heterozygosity (HA), Polymorphism Information Content (PIC) at different microsatellites loci in Diara buffalo population were obtained 7.4, 5.7, 1.8, 0.68, 0.82, 0.80 and 0.79, respectively. The allele distribution followed the normal L-shaped form suggesting that the breed had not encountered a genetic bottleneck in the recent past. The result shows sufficiently high genetic diversity in the population. The information generated in this study may aid in formulation of effective breeding and conservation programme. However, genetic diversity study of Diara buffalo needs to be extended to include more microsatellites in a large sample size to further validate the research.