Characterization of genetic diversity by employing molecular tools is a prerequisite in developing strategies for conservation and utilization of poultry genetic resources. We assessed genetic variation within and between six Indian chicken populations (Naked Neck, White Silkies, Commercial Layer and Broiler, Giriraja and Desi) using nine microsatellite markers. Seven loci were polymorphic in all populations, with the number of alleles varying from 3 to 27per locus and 4.1 to 8.6per population. Average heterozygosity and gene diversity in the populations were 0.68 and 0.66, respectively. Some populations and loci deviated from Hardy-Weinberg equilibrium. Except Naked Neck, none of other populations was in genotypic disequilibrium for a single pairwise locus combination. F-statistics indicated that populations are genetically structured, ∼85% of the total genetic variation being due to genetic differentiation within the populations. Estimates of Latter’s FST and Nei’s DS genetic distances revealed relatively close genetic similarity between Naked Neck and Giriraja, while White Silkies was the most distant. An estimated divergence time between Naked Neck and Giriraja was over100 years, with a comparatively longer (over 450 years) period of separation between Giriraja and White Silkies. We suggest that there is an essential room left for genetic improvement of Indian native breeds and commercial stocks.