In this study, twelve microsatellite loci were utilized to determine genetic differences between five Egyptian buffalo populations and genetic characterization of these populations. A total of 80 blood samples were collected randomly from five farms in five different governorates (Cairo, Kafr El-Sheikh, Menoufia, Ismailia, and Beni Suef). The genetic parameters like number of alleles, allelic frequencies, F parameters inbreeding coefficient (F_IS), Total inbreeding coefficient (F_IT) and Population differentiation (F_ST), deviation from Hardy-Weinberg Equilibrium (HWE), genetic distances and evolutionary relationships between pairs of studied buffalo populations were obtained using four different programs. Results revealed that all the five populations under study had significant deviations from HWE, as well all loci deviated significantly from HWE across all populations except locus BM1818. This is attributable to non-random mating and selection within these populations, presence of null alleles at some loci, and transfer of buffaloes from one place to another which led to underestimation of heterozygosity. Values of F_IT across all populations were high, whereas, values of F_ST were low, indicating the high degree of inbreeding in these populations and the low degree of genetic differentiation among them. The lowest genetic distance was found between Sids and Ismailia populations (0.200) while the highest genetic distance was detected between Kafr El-Sheikh and Ismailia (0.373). Genetic distances and neighbor joining showed that Sids and Ismailia populations were grouped together than the remaining groups (Cairo, Kafr El-Sheikh, and Shebeen El-Kom populations). The information obtained by this study will assist in the establishing effective breeding programs and furthermore, in conserving indigenous Egyptian buffalo breed in the future