Abstract. Investigation of genetic relationship among populations has been traditionally based on the analysis of allele frequencies at different loci. The prime objective of this research was to measure the genetic polymorphism of five microsatellite markers (McMA2, BM6444, McMA26, HSC, and OarHH35) and study genetic diversity of 14 sheep types in Iran. Genomic DNA was extracted from blood samples of 565 individuals using an optimized salting-out DNA extraction procedure. The polymerase chain reaction (PCR) was successfully performed with the specific primers. Some locus–population combinations were not at Hardy–Weinberg equilibrium (P < 0. 05). The microsatellite analysis revealed high allelic and gene diversity in all 14 breeds. Pakistani and Arabi breeds showed the highest mean number of alleles (11.8 and 11 respectively), while the highest value for polymorphic information content was observed for the Arabi breed (0.88). A UPGMA (unweighted pair group method with arithmetic mean) dendrogram based on the Nei's standard genetic distance among studied breeds showed a separate cluster for Arabi and Pakistani breeds and another cluster for other breeds. The Shannon index (H0) for McMA2, BM6444, McMA26, HSC, and OarHH35 was 2.31, 2.17, 2.27, 2.04 and 2.18, respectively, and polymorphic information content (PIC) values were 0.88, 0.92, 0.87, 0.84, and 0.86 for McMA2, BM6444, McMA26, HSC, and OarHH35, respectively. The high degree of variability demonstrated within the studied sheep types implies that these populations are rich reservoirs of genetic diversity that must be preserved.