One of the traits of the greatest financial impact on sheep production is the number of lambs weaned per ewe with FecB considered as the most well known major gene concerned with this trait. In order to conduct a marker assisted introgression (MAI) program the presence of FecB responsible mutation in Afshari sheep breeding flock was surveyed. Direct test to detect FecBB allele was conducted on 74 DNA samples through 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 that did or did not carry introgressed gene. Except for MCMA26 monomorphic locus, other microsatellite loci exhibited a moderate level of polymorphism. As such a total of 102 alleles were detected with a mean number of 6 alleles per locus. A full characteristics assay of this set of seventeen polymorphic loci was carried out, generating allele frequency distributions that were employed 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 were 067 (SD=0.08). Cumulative value of I corrected for linked loci was estimated as 3.128E-13. The results of microsatellite analysis indicated that, despite the selective breeding along with closed flock system over a number of generations, a relatively high level of allelic variability still exists in the breeding flock. Besides, the results of banding patterns from AvaII digestion of PCR products indicate the absence of FecB mutation in this elite flock. These observations demonstrate the applicability of marker data and would assist in making decisions on starting MAI program for an inclusion of FecB gene into the Afshari breed.