Objective To compare and analyze the genetic structure of NIH mice bred in Unites A and B, using microsatellite technology. Methods Thirty SPF 8-week old outbred NIH mice (half male and half female) of each population were randomly chosen from the Units A and B, respectively. PCR amplification and STR scan were performed to determine the genetic characteristics of two outbred populations using microsatellite loci, and the population genetic structure was analyzed with statistical software Popgene 1.32. Results In the NIH mouse population form the Unit A, 74 alleles were obtained, with an average heterozygosity of 0.3108 and polymorphism information content of 0.2637. In the NIH mouse population from the Unit B, 76 alleles were obtained, with an average heterozygosity of 0.3257 and polymorphism information content of 0.2777. The inter-population comparison showed that genetic differentiation coefficient Fst was 0.3932, the genetic identity was 0.3971, and the genetic distance was 0.9235. The population difference was significant. Conclusions There is serious genetic differentiation between the two NIH mice populations, resulting in the formation of two different closed populations.