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The genetic diversity and population structures of five Chongqing local chicken populations were investigated using by 24 microsatellite markers. Results revealed that the mean number of alleles (NA) ranged from 7.08 (Daninghe chicken, DN) to 8.46 (Nanchuan chicken, NC). The highest observed heterozygosity (HO) and expected heterozygosity (HE) were observed in DN (HO = 0.7252; HE = 0.7409) and the lowest HO and HE were observed in XS (Xiushan native chicken [XS], HO = 0.5910 and HE = 0.6697). The inbreeding coefficient (FIS) within population ranged from 0.022 (DN) to 0.119 (XS). Among the 24 microsatellite markers, four loci (MCW0111, MCW0016, ADL0278, and MCW0104) deviated from the Hardy–Weinberg equilibrium in all the studied populations. The results of population polygenetic analysis based on Nei’s genetic distance and STRUCTURE software showed that the clustering of the five populations was incomplete consistent with geographical distribution. Moreover, a large number of gene flows were widespread among different populations, suggesting that genetic material exchanges occurred due to human activities and migration which was also verified by PCoA. In summary, this study preliminarily showed that Chongqing local chicken populations had rich genetic diversity and remarkable genetic divergence, but still high risk in conversion. These findings would be useful to the management of conservation strategies and the utilization of local chicken populations in further.