Background Genetic diversity of sheep in Jordan was investigated using microsatellite markers (MS). Six ovine and bovine MS located on chromosomes 2 and 6 of sheep genome were genotyped on 294 individual from ten geographical regions. Results The number of alleles per locus (A), the expected heterozygosity (He) and observed heterozygosity (Ho) were measured. Overall A, He and Ho were 12.67, 0.820 and 0.684, respectively. On the other hand, genetic distances undoubtedly revealed the expected degree of differentiation among the studied populations. The finding showed closeness of three populations from south (Maan, Showbak and Tafeilah) to each other. Populations from the middle regions of Jordan (Karak, Madaba, Amman, AzZarqa and Mafraq) were found to be in one cluster. Only two populations of the middle region were an exception: AlSalt and Dead Sea. Finally, sheep populations from Irbid were located in separated cluster. It was clear that the studied predefined populations were subdivided from four populations and would be most probably accounted as ancestral populations. These results indicate that number of population is less than the predefined population as ten based on geographical sampling areas. Conclusions The possible inference might be that geographical location, genetic migration, similar selection forces, and common ancestor account for population admixture and subdivision of Awassi sheep breed in Jordan. Finally, the present study sheds new light on the molecular and population genetics of Awassi sheep from different regions of Jordan and to utilize the possible findings for future management of genetic conservation under conditions of climate changes and crossbreeding policy.