Objective To conduct a comparative study of the intestinal flora in six common strains of mice and rats. Methods In this study, 210 feces or intestinal content samples were collected from 150 SPF mice and rats from three experimental animal production units. PCR amplification of the samples was carried out based on common primers of the bacterial 16S rDNA V3-V4 region, and then the Illumina HiSeq 2500 high-throughput system was used for sequencing. Based on the animal source, breed, gender, age and intestinal segmentation ( duodenum, jejunum, ileum, cecum, colon and rectum) factors, bioinformatics method and cluster analysis of differences were applied to analyze the diversity in the structure of the intestinal flora in rats and mice. Results The intestinal flora of experimental mice or rats with the same source had the highest similarity, whereas the intestinal flora of experimental mice and rats showed certain differences due to the type of species. The intestinal flora from different intestinal segments of experimental mice exhibited certain differences, and stool samples were more similar to the colonic intestinal flora. Under the same breeding and living environment conditions, the species, age and intestinal segment of experimental mice and rats are the main endogenous factors that determine the structure of the intestinal flora, and the effect of strain and gender on their intestinal flora is minimal. Conclusions This study revealed the diversity and differences in the intestinal flora of common mice, explored the influencing factors of the host intestinal flora, and provided more basic data for the relationship between intestinal flora and diseases.