Abstract: The traits with the greatest financial impact on sheep production are the number of lambs weaned per ewe and growth efficiency. FecB and Callipyge are the most well known major genes concerned with these traits. In order to conduct a Marker Assisted Introgression (MAI) program we surveyed the presence of responsible mutations in Afshari sheep breeding flock. Direct tests to detect of FecBB and CLPG alleles were conducted on 74 and 58 DNA samples respectively by PCR-RFLP assay. Also the polymorphism content of the breeding flock was investigated by genotyping of one hundred randomly sampled animals at eighteen microsatellite loci selected on the chromosomes carrying and not carrying introgressed genes. A part from MCMA26 monomorphic pattern, microsatellite loci showed moderate level of polymorphism, as such totally 102 alleles were detected with a mean number of 6 alleles per locus. A full characterization of this set of seventeen polymorphic loci was carried out generating allele frequency distributions that were used to estimate genetic parameters of these loci, including expected heterozygosity, Polymorphism Information Content (PIC), probability of identity (I) and discrimination power (D). The average expected heterozygosity was 0.72 (SD = 0.07) and the average PIC was 0.67 (SD = 0.08). Cumulative value of I considering for linked loci were estimated as 3.128E-13. The results of microsatellite analysis indicated that, despite the selective breeding and closed flock system over a number of generations, a relatively high level of heterozygosity still exists in the breeding flock. Besides, the banding patterns resulted from AvaII digestion of FecB and CLPG amplicons approved the absence of the mutations in this flock. These observations demonstrate applicability of marker data and would assist to make decision on starting MAI program for inclusion of FecB and CLPG genes into the Afshari breed.