Objective To study the genetic features of different populations of Kunming mice in order to enrich their genetic data and explore the method for genetic quality control. Methods SPF KM mice from two populations of Liaoning province (30 mice in each group, male:female=1:1) were used in this study. Ten microsatellite loci on different chromosomes with rich polymorphism information were chosen, and the heterozygosity (h_e), polymorphism information content (PIC) and genetic distance (D_A) were analyzed by PCR amplification and PAGE electrophoresis. Eleven morphological parameters of the mandible were determined. Results Twenty-eight alleles and 31 genotypes were found in the two mouse populations, with a h_e of 0.472 and PIC of 0.382. Twenty-seven alleles and 28 genotypes were found in the population A, with a h_e of 0.525 and PIC of 0.402. Twenty-six alleles and 27 genotypes were found in the population B, with a h_e of 0.418 and PIC of 0.361. D_A between the two populations was 0.076. The genetic diversity of group A was slightly higher than that of the group B. There were some differences in the mandibular morphology between the two populations, among them, 5 parameters had significant differences (P<0.05 or P<0.01). Conclusion The genetic distance of the two Kunming mouse populations is approximate, with a weak genetic differentiation between the two populations.