Objective: To analyze the intestinal microbial composition in specific pathogen free(SPF) mice using high-throughput sequencing. Methods: A total of 65 SPF mice of 14 strains were collected. Samples of fresh ileocecus content from the SPF mice were examined using high-throughput sequencing, and 16S rRNA V3-V4 variable regions of bacterium were deeply sequenced. The composition, alpha diversity, and the abundance of microbial flora in ileocecus content were explored, while Staphylococcus, Pseudomonas, Mycoplasma and Corynebacterium were identified at the genus level. Results: Altogether 144 318 high-quality genome sequences were obtained after data filter and quality control. Remarkable differences were found in Alpha diversity (species richness, diversity and evenness) of the microbial composition between samples from mice of the same strains. At the phylum level, the main sequences were largely concentrated as follows: Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Actinobacteria, Saccharibacteria, Tenericutes and Spirochaetae. At the genus level, Staphylococcus, Pseudomonas, Anaeroplasma and Corynebacterium were detected in some samples. Conclusions: The composition, alpha diversity and the abundance of the microbial flora in ileocecus contents are substantially different in mice of same strain. There is a need to keep intestinal microbial composition in SPF mice under control, and high-throughput sequencing may offer such possibility.