BACKGROUND: Understanding the genetic background of Korean field mouse(Apodemus peninsulae Thomas, 1906) is important for employing the animal as an experimental model in research. However, limited genetic information is available about A. peninsulae. METHODS AND RESULTS: This study aimed to develop microsatellite molecular markers based on the genome sequence of A. peninsulae and establish a genetic evaluation system for A. peninsulae. Twenty-nine polymorphic microsatellite markers were identified via electrophoretic analysis, short tandem repeat scanning, and sequencing, and genetic diversity in three populations of A. peninsulae from Dalian City, Liaoning Province (LN), Changchun City, Jilin Province (JL) and Mudanjiang City, Heilongjiang Province (HLJ) was analyzed. A total of 229 alleles (Na) were detected in 115 A. peninsulae individuals. The mean observed alleles and effective alleles (Ne) were 7.897 and 3.571, respectively. The Shannon index(I) averaged 1.401, indicating high genetic diversity, whereas the mean observed heterozygosity (Ho) and expected heterozygosity (He) were 0.543 and 0.668, respectively. The mean polymorphism information content (PIC) was 0.627, which validated high genetic diversity in the three populations. Analysis of molecular variance (AMOVA) (genetic differentiation coefficient (FST) = 0.062) showed that the genetic distances were 0.083 (LN and JL), 0.203 (LN and HLJ), and 0.195 (JL and HLJ), indicating that the two artificially domesticated populations were genetically indistinguishable. Meanwhile, the wild HLJ population was significantly different from the other two artificially domesticated populations. The results of structural analysis, phylogenetic tree construction, and principal component analysis (PCA) were consistent with those of AMOVA. In addition, gene flow analysis used to explore genetic exchange confirmed the flow of genetic information among the three populations. CONCLUSION: The microsatellite loci identified in this study are highly polymorphic and suitable for the genetic quality control of A. peninsulae, providing an important genetic basis for the breeding of A. peninsulae and evaluating its genetic potential. Thus, this study lays a scientific foundation for the future genetic improvement and resource utilization of this species.