Objective The genetic structure composition of the ddY mouse population, which was bred in isolation for a long time, was compared with that of the newly introduced ddY mouse population by microsatellite analysis. Methods Thirty-six microsatellite loci were analyzed. Thirty-eight DNA samples were randomly collected from each of the two groups. The DNA samples were then amplified by PCR and the PCR products were sequenced. PopGen1. 32 and Littleprogram0. 6 were used to process the data. Results The average allele number of group A was 2. 4839, the average heterozygosity was 0. 3376, and the average polymorphism information content was 0. 2875. Group B had an average allele number of 3. 4839, average heterozygosity of 0. 4806, and an average polymorphism information content of 0. 4088. Conclusions The genetic structure of the two laboratory mouse populations was different, and closed breeding result ed in genetic structure changes in the populations. It is necessary to monitor the genetic structure of closed laboratory animal populations regularly to ensure the genetic quality of populations.