The present study aimed to outline the population differentiation of Sinai and Norfa chicken, native to Egypt, with microsatellite markers. Twenty microsatellite loci recommended by the Food and Agriculture Organization (FAO) were used. Fifty eight birds were sampled (29 for each strain: 12 males + 17 females). Data were collected and genetic diversity indicators were assessed utilizing the approaches implemented in FSTAT, Cervus 3.0.7 and GenAlEx 6.5 software programmes. A total number of 182 alleles were detected with an average value of 9.1 allele per locus. The expected heterozygosity was 6.625 and 6.343 in Norfa and Sinai chickens, respectively. Norfa chickens produced 15 private alleles, while there were 9 unique alleles detected in Sinai chickens (13.18% private alleles as a percentage of the total observed number of alleles). Fixation indices’ (FST, FIS, and FIT) values were 0.060, 0.410 and 0.438, respectively, across all 20 loci investigated. Results indicated that the studied populations were genetically differentiated. Consequently, they have high breeding potential. Efforts should be made to incorporate the other local chicken strains as unique genetic resources into conservation programmes. This should begin with proper management of these flocks to ensure the maintenance of their genetic diversity over time by avoiding inbreeding. Such information is likely to have a profound effect on the success of genetic improvement and completes information from phenotypes and biometric measurements of the domestic chickens in Egypt.