Objective The intestinal flora is closely related to nutritional obesity. Most of the previous research has focused on the fecal flora of the large intestine, with very few studies on the flora of the small intestine, which is the main site of absorption. We aimed to investigate the differences in bacterial flora between the colon and the ileum in a rat model of nutritional obesity using microbial diversity analysis. Methods Male Sprague-Dawley (SD) rats were fed with a high-fat diet (HFD) mixed with chow for 60 days to establish a nutritional obesity model, then fed with chow for another 60 days to eliminate the influence of the HFD. Total DNA was extracted from the contents of the ileum and colon and used as template for amplification of the V3 + V4 region of the 16S rRNA gene by PCR, then sequenced to establish operational taxonomic units (OTUs). Annotation and taxonomic analysis were performed against the Silva database. Results (1) Alpha analysis showed that the number of intestinal flora was higher than that of colon ( chao1, abundance-based coverage estimator), but the diversity was lower than that of the colon ( simpson’ s Index, shannon function) (P < 0. 05); (2) Beta analysis showed that the species similarity between the ileum and colon + ileum samples was low; (3) OTU annotation and cluster analysis showed that there were obvious differences in the types and species abundance distributions of dominant bacteria (top 10 in abundance) in the ileum and colon, with low overlap at the genus level; (4) The abundance of Rothia in the ileum of HFD-obesity resistant rats increased significantly, while that of Romboutsia decreased. Conclusions The ileum and colon had different levels of diversity in intestinal flora in rats with nutritional obesity. Rothia and Romboutsia may be the key ileum flora involved in obesity.