Abstract: In order to build a mouse（Mus musculus） population model of NOD/ShiLtJ-C57BL/6J recombinant inbred（RI） strain（NXB RI for short）, to ensure the balance of parental gene coverage and to improve inbreeding efficiency, this study used genotyping by sequencing（GBS） technology to genotype and sequence 200 F5 NXB RI strains, and then principal component analysis, genetic differen-tiation coefficient and genetic distance analysis and kinship analysis among individuals were used to identify the genotype coverage and inbreeding degree of mouse population parents. By sequencing 200 samples from 36 subgroups of F5 NXB RI strains, a total of 115 634 single nucleotide polymorphism（SNP） were measured, of which 41.67% were C57BL/6J homozygous sites, 27.05% were NOD/ShiLtJ homozygous sites, 26.79% were heterozygous sites, and the SNP sites were distributed in all chromosomes; 94.6% of the subgroups had F_st values greater than 0.15, indicating that the genetic structure of most F5 NXB RI strains differed greatly and the degree of differentiation was high. The mean value of identity by state（IBS） distance among the 200 NXB RI samples was 0.667, the highest was 0.925, and the lowest was 0.480, which could provide basis for next generation breeding screening. The data of hearing phenotype and fasting blood glucose phenotype of F5 NXB RI strains followed normal distribution. The results of the above studies showed that F5 NXB RI strains had a relatively balanced distribution of parental genotype coverage, and could retain more NXB RI strains. The resulting NXB RI strains, as a new generation of genetic reference population, can be used as a research model for related complex genetic diseases.