Background: The development of local sheep breeds that have high fecundity levels, high meat content and high growth rates would help satisfy the Palestinian demand of meat and lower its price. It has been reported that mutant alleles of the genes Booroola fecundity (FecB), Callipyge (CLPG) and calpastatin (CAST) are associated with high litter size, larger muscular rumps and higher tenderness of meat after sheep slaughter, respectively. PCR-RFLPs methods have been established to facilitate the survey of sheep breeds for such mutants.Methods: In this study, 117 blood samples of the most common sheep breed in Palestine, Assaf, were collected from Bethlehem and Jenin districts. Genomic DNA was extracted and purified from the blood samples using the salting out method. The genotypic ratios and allelic frequencies of the FecB, CLPG and CAST genes were determined by PCR-RFLP method. Result: The results show that the mutant allele B of the FecB gene has a very low frequency among the screened sheep with 0.01 in Bethlehem and 0.07 in Jenin. Also, 94.4% of the tested sheep have the wildtype genotype AA for the CLPG gene with a frequency of 0.95 for allel A. Moreover, the desired heterzygous genotype MN for the CAST gene is 38.4% with an allelic frequency of 0.81 for the mutant allel M. These results would assist establishing sheep breeding programs with the appropriate parental genotypes to improve the livestock sector in the country.