Objective To study the diversity of intestinal flora in commonly used SPF mice and rats. MethodsThe cecum contents of C57BL/6, ICR, BALB/ c mice as well as Wistar and SD rats were collected from three experimentalanimal production units in the Guangdong area. The V4-V5 region was amplified using 16S rDNA primers and sequenced bythe Illumina Miseq 2x 300 bp sequencing platform. Microbial community analysis, alpha diversity analysis, and betadiversity analysis were carried out by bioinformatics. Results In OTU cluster analysis after sequence removal optimization,the dilution curve indicated that the data quantity of the sequencing was reasonable. The intestinal microflora of mice andrats were divided into eight phyla. Bacteroidetes and Firmicute phyla occupied the main position, mainly in the level ofBacteroides, followed by the genuses Hungatella and Parabacteroides and then Lactobacillus. Analysis of the differencesamong the samples showed that the flora composition of animals from the same facility, and that of the same strains from thesame facility was also similar. Alpha analysis showed that the species richness was similar in animals from the same facility,and beta analysis showed little difference in intestinal flora of the same origin animals. Moreover, the strain had an effect onthe difference of intestinal flora. Conclusions The origin is the main factor influencing the diversity of intestinal flora, and the strain has a certain influence on their diversity.