Objective To explore the objectivity and scientificity of fecal sampling, and to provide reference for investigating the relationship between intestinal microbes and diseases. Methods Terminal restriction fragment length polymorphism, degeneration gradient gel electrophoresis and real time fluorescent quantitative PCR techniques were applied to differentially analyze the bacterial community composition and abundance of intestinal contents and feces taken from different sites of BALB/c mouse intestine. Results The predominant T-RFLP fragments (T-RFs) in feces in the rectum and colon were 244 bp, 255 bp and 449 bp, however, those in feces of the small intestine including duodenum, jejunum and ileum were 60 bp, 73 bp, 261 bp, 268 bp and 272 bp, and with a larger variation of the bacterial community composition in various parts of the small intestine. The bacterial abundance in the contents of duodenum and jejunum were 6.9 log (copies)/g and 8.3 log (copies)/g, fewer than in the other parts of the intestine, while the bacterial abundance in the feces was as high as 11.8 log (copies)/g, being about 2 times higher than that in the duodenum and jejunum (P<0.05), and similar to that in the ileum and colon content (P>0.05). Conclusions The inter-mouse variations of bacterial community composition in the large intestine contents are small. The bacterial composition and abundance are similar suggest that studies on the relationship between large intestine especially colorectal microbiota and diseases may be conducted via fecal sampling.