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Indian Aseel chicken (Gallus gallus) is traditionally used as a favorite game bird all over the world. Bird fighting communities of Pakistan are the major source of its conservation and there are at least four distinctively recognized varieties of Aseel chicken based upon selective breeding, geographical location and color patterns. A pioneering study on genetic diversity of these varieties namely Lakha (n=17), Mushki (n=19), Mianwali (n=19) and Peshawari (n=13) was undertaken using FAO recommended 10 microsatellite loci. A total of 91 alleles were observed in 4 varieties of Aseel chicken with an average of 9.1 alleles per locus. Number of alleles varied between 4 to 8 in Lakha, 4 to 9 in Mushki, 3 to 10 in Mianwali and 3 to 7 in Pashawari. Mean polymorphic information content values were 0.67, 0.69, 0.71 and 0.65 in individual varieties, respectively. Mean observed and expected heterozygosity index values of 0.3941 and 0.7376 were recorded in Lakha, 0.4105 and 0.7468 for Mushki, 0.4105 and 0.7718 Mianwali and 0.3692 and 0.7191 for Peshawari. Mean Fixation index (Fst) value was calculated as 0.1264. Highest Nei’s standard genetic distance (Ds) value of 1.0735 was observed between Mushki and Peshawari, whereas its value was minimum (0.3533) between Lakha and Mushki. This report describes genetic diversity of Aseel chicken in Pakistan and provides foundation data to initiate extensive and more comprehensive studies on indigenous chicken genetic resource conservation and its future utilization in commercial breeding programs.