Bacterial populations in the ileum of broiler chickens were analyzed by molecular analysis of 16S rRNA genes and compared to those in the cecum. Bacteria found in the ileal mucosa were mainly Gram-positive with low G+C content. There were 15 molecular species among 51 cloned sequences. More than 70% of the cloned sequences were related to lactobacilli and Enterococcus cecorum. Two sequences had 95% or less homology to existing database sequences. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed differences among bacterial populations present in the mucosa and lumen of the ileum. Comparative studies by T-RFLP and sequence analyses of 16S rRNA genes indicated a less diverse bacterial population in the ileum (mucosa and lumen) than in the cecum. Lactobacilli, E. cecorum, and butyrate-producing bacteria related (including both identified and unidentified species) sequences were the three major groups detected in ilea and ceca. Although butyrate-producing bacteria may have good potential in the development of novel probiotics for poultry, verifying the presence of the bacteria in the chicken gut is required to warrant further investigation.