Sixty four blood samples were collected from unrelated buffaloes from different populations representing three suspected breeds (Menofi, Beheri and Saidi). The first population was the middle Delta region included: Kafr El-Sheikh and Menoufia; the second was Alexandria population included Alexandria only, while the third population was Upper Egypt (from Assuite up to Sohaj). Five bovine microsatellites (HEL013; CSSM066; ILSTS005; INRA035 and HEL001) were analyzed in the three different populations of Egyptian buffalo in order to determine the genetic diversity between and within populations as well as assess the purity or the heterogenecity of each population. The tested microsatellites showed polymorphism in all populations meaning that they could be used further in parentage testing as well as mapping of quantitative trait loci (QTLs) in buffalo. Contrarily, the microsatellite HEL001 was monomorphic in the studied populations. A total of 29 alleles were detected across the microsatellite loci when they were screened in all populations. The mean number of alleles per locus was 7.25 alleles. However, the observed number of polymorphic alleles ranged from five (ILSTS005) to ten (HEL013). Heterozygosity and Wright's F-statistics (FIS, FST, and FIT) were calculated to determine the genetic variation in these populations. High values of observed heterozygosities were noticed in all populations, the lowest heterozygosity value was 0.333 which observed in Upper Egypt for ILSTS005. In contrast, a complete heterozygosity value (1.00) was observed in many populations, since it observed in Upper Egypt for HEL013 and INRA035; in Alexandria for HEL013; CSSM066; INRA035 and also was observed  in Delta for CSSM066 and HEL013. All FIS values were below the zero, meaning the absence of inbreeding within each population. High averages of gene diversity were noticed for all markers and all populations, ranging from 0.665 to 0.753 with an overall mean of 0.709. Values of gene flow or migration between populations were high meaning that migration and admixture could have taken place between these populations. Depending on the genetic distances, a dendrogram showing the genetic relationship among the different populations.