As the progress of molecular genetics techniques, it is now possible to determine the relationship between individuals and their evolutionary process. In this study, the genetic structure and assignment test of Kurdish horses breed to its origin or Arab and Turkmen horses breed were investigated using 12 microsatellite markers recommended by the International Society for Animal Genetics (ISAG). For this purpose, blood samples from 238 Kurd, 36 Arab and 30 Turkmen horses were taken in their distribution areas. DNA was extracted from Blood samples by the salting-out method. DNA fragments were amplified by multiplex PCR reaction using fluorescently labeled primers and determined by capillary electrophoresis. The mean of observed and effective alleles for all markers was 9. 9 and 4. 71, respectively. The highest and lowest expected heterozygosity was obtained for ASB17 (0. 83) and HTG4 (0. 71) markers, respectively. Cluster analysis using the UPGMA method and the Dice coefficient differentiated each breed. The result of the Bayesian Assignment test showed that all individuals correctly assigned to its population, so it seems that these markers are useful for the assignment test. Overall, the results of this study showed that the microsatellite markers used in this study showed high genetic diversity, so the use of these markers for genetic diversity study and assignment test of the Kurdish horse is recommended. So, these markers can be used to determine the exact origin of the Kurdish horse breed and eliminated the non-purebred horses from the reproductive process.