Objective To analyze and evaluate the population genetic quality of outbred KM mice from National Rodent Seed Center (Shanghai). Methods A total of 30 outbred KM mice were randomly chosen. The genetic characteristics of the population were determined by PCR and STR scanning using 30 selected microsatellite loci. Popgen1.32 software was used to process the data.Results Thirty microsatellite loci shared 123 alleles in the KM mouse population. The average effective allele number and the average heterozygosity were 2.3989 and 0.5342, respectively. The average polymorphism information content (PIC) was 0.4735. Conclusions The outbred KM mouse population of Shanghai Seed Center has genetic stability and genetic diversity, and is satisfied with the genetic characteristics of closed colony laboratory animal.