Objective To analyse and monitor the genetic quality of closed colony NIH mice in Beijing district for the last 3 years. Methods We use biochemical genetic markers(including alkaline phosphatase-1 and the like 14 biochemical markers), selecting A and B colonies from different facilities for genetic monitoring in 2011 to study the polymorphism. And in 2014, 30 NIH mice just from B colony were monitored using the same testing and sampling methods. Results In 2011, NIH mice form both A and B facilities existed 6 polymorphic biochemical markers(Ce2, Car2, Gpi1, Es10, Gpd1, Pgm1); and NIH mice of B company also existed polymorphism in Es3 loucs. Between the 2 NIH mice colonies, there were significant difference in Es3、Gpd1、Pgm1 loci (P < 0.01), and difference in Car2 locus(P < 0.05). FST of the 2 colonies was 0.0406, the genetic identity was 0.9619, and the genetic distance was 0.0388. In B company, NIH mice of 2014 appeared 2 homozygous loci(Ce2 and Gpd1) when compared with NIH mice of 2011. Between the 2 NIH mice colonies, there were significant difference in Es10 and Gpd1 loci (P < 0.01), and difference in Pgm1 locus(P < 0.05). Fst of the 2 colonies was 0.1103, the genetic identity was 0.8847, and the Genetic distance was 0.1266. Conclusions Population isolation, breeding and selection, populationpopulation quantityquantity and generation significantly affected the genetic architecture of NIH mice. So when breeding and reserving seeds, we should strengthen the genetic monitoring of outbred NIH mice, in order to offer reliable genetic quality protection.