The present study was conducted to know the genetic diversity of three Saudi sheep populations; Naeimi (NM), Herri (H) and Najdi (NJ). Genomic DNA was extracted from 156 animals of sheep comprising 47 Naeimi, 47 Herri and 62 Najdi breeds using 18 microsatellite markers. A total of 212 alleles were generated with a mean value of 11.80 alleles per locus, with a range of observed and expected heterozygosity of 0.505 to 0.875 and 0.595 to 0.854, respectively. Eleven of the microsatellites loci studied in NM, three loci in H and fifteen loci in NJ were observed to be deviated from Hardy-Weinberg equilibrium. The fixation genetic indices (Fst) among the three sheep populations were very low, ranging from 0.017 (between NJ and H) to 0.033 (between NJ and NM), indicating low population differentiation among the three sheep populations studied. The present study showed that the microsatellite markers are powerful tool in determining genetic diversity among sheep populations.