A total of 210 chicken samples, from seven strains, were genotyped using 20 microsatellite loci of which 16 are recommended by the Food and Agriculture Organization. The genetic variability and divergence of four Thai indigenous strains and three commercial lines were assessed to generate baseline information for conservation, promotion, and make sustainable utilization of indigenous chicken resources in Thailand. A total of 227 alleles were distributed ranging from six (MCW 111) to 16 (MCW 183 and LEI 166) alleles per locus. The highest (0.81) and lowest (0.77) average of expected heterozygosities were observed in indigenous chicken (Dang) and commercial layer (Isa Brown), respectively. All microsatellite loci were in the Hardy-Weinberg equilibrium, except for MCW111 and ADL372 in the Isa Brown line. The subpopulation division coefficient (F(ST)) was strong with the value of 0.183 indicating the genetic differentiation among the studied groups. Four genetic clusters were detected: the first group consisted of layers (Isa Brown and White Leghorn); the second group was broiler; the third group consisted of non-black feather indigenous chicken (Chee, Dang, and Leung Hang Khoa); and the fourth group was black feather indigenous chicken (Pradu Hang Dam). The results of this study also suggested that Pradu Hang Dam is suitable to be developed as a meat type chicken due to lower genetic distance between Pradu Hang Dam and broiler.