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In order to analyze population genetic structure at multiple spatial scales,microsatellite loci were developed for the ornamented pygmy frog (Microhyla fissipes),and 15 polymorphic microsatellite loci were successfully screened from 105 individuals,of which 82 from four populations distributed in the Sichuan Basin and 23 from Sangzhi population in western Hunan.Five loci were found to significantly depart from Hardy-Weinberg equilibrium in one to three populations,probably due to small sample size.No significant linkage disequilibrium was detected after sequential Bonferroni corrections.The average number of alleles in all loci was 8.5,ranging from 0.63 to 0.90,respectively.The average polymorphism information content was 0.678,proving that the genetic diversity of M.fissipes was high.The Sangzhi population and the remaining four populations can be clearly separated using Bayesian clustering methods,showing that the species M.fissipes was probably affected by the topography,especially mountain barriers.These polymorphic microsatellite loci could be used for further study on the landscape genetics of this highly adaptive and widely distributed species.