Objective To analyze the population genetic quality of the same closed KM mouse colony in 2013 and 2020 by microsatellites. Methods DNA was extracted from 30 KM mice in 2013 and 2020. Thirty pairs of microsatellites primers were used for PCR amplification and DNA sequencing to evaluate genetic quality by the number of alleles, heterozygosity and polymorphism content. Results Ninety-five alleles were found in 2013, average heterozygosity was 0. 4864 and average polymorphism content was 0. 4418. There were 122 alleles, average heterozygosity was 0. 5150, and average polymorphism content was 0. 4818. Conclusions The closed KM mice colony had genetic stability and genetic diversity, which satisfied the genetic characteristics of a closed colony of laboratory animals.