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The study aimed to research the genetic variation of the Awassi and Naimi sheep breeds using the two genes myostatin (MSTN) and calpastatin (CAST). Blood samples were collected from 100 animals of the two breeds, and then DNA was extracted using a commercial kit. We used the PCR and RFLP techniques to determine genotypes and allele frequencies. The results showed that the MSTN and CAST genes are polymorphic. The MSTN gene has allelic frequencies (M and m) of 0.81, 0.19, and 0.76, 0.24 in the Awassi and Naimi breeds, respectively. The frequencies of the genotypes MM, Mm, and mm in the Awassi breed were 0.70, 0.19, and 0.11, but in the Naimi breed, they were 0.67, 0.13, and 0.20, respectively. Moreover, the number of alleles observed (Na), the effective number of alleles (Ne) and observed (Ho), and expected (He) heterozygosity were found to be 3, 2.30, 0.24, and 0.35 in the Awassi breed and 2, 1.62, 0.17, and 0.26 in the Naimi breed, respectively. The allelic frequencies (M and N) of the CAST of the Awassi and Naimi breeds are 0.86, 0.14, and 0.88, 0.12, respectively. The frequencies of the genotypes MM, MN, and NN in the Awassi breed were 0.94, 0.04, and 0.02, respectively, while for the Naimi breed, they were 0.95, 0.02, and 0.03, respectively. Also, the Na, Ne, Ho, and He were found to be 2.8, 1.72, 29.6, and 28.57 in the Awassi breed and 1.10, 1.23, 0.17, and 0.15 in the Naimi breed, respectively. According to the chi-square of MSTN and CAST genes, both breeds were not in Hardy-Weinberg equilibrium balance.