Abstract In the present study genetic variation within three Kurdish horse populations (Kurdi, Kalhor and Afshari) and between three Iranian horse breeds (Arabian, Kurdish and Arab-Kurd) was assessed using two microsatellite markers (NVHEQ70 and HMS7). All loci were polymorphic in all populations. All locus-population combinations were at Hardy-Weinberg nonequilibrium (p<0.05). The lowest genetic distance (0.073) was obtained between Kurdish and Arab-Kurd and the highest genetic distance observed (0.243) between Kurdish and Arabian. Within Kurdish population, the lowest and the highest genetic distance were observed between Afshari and Kalhor and between Kurdi and Afshari, respectively. The dandrogram based on genetic distance was drown using unweighted pair group method using an arithmetic average (UPGMA). The Kurd and Arab-Kurd were located together at one cluster and Arabian at another. In Kurdish population, Afshari and Kalhor were located at one cluster and Kurdi at another.The average expected heterozygosity for each breeds ranged from 0.227 to 0.844 for Turkaman and Kurd, respectively. In Kurdish population, the small difrences expected heterozygosity obtained between highest and lowest. All markers have relatively high polymorphic information content (PIC) value (> 0.6). One the base of finding of this research, we can conclude that microsatellite variation could be an useful tool for screening of biodiversity domestic animal. Keyword: Kurdish horse, Microsatellites markers, Genetic variation, Heterozygosity, Polymorphism