The gastrointestinal tracts of broiler chickens contain complex microbial communities that influence numerous aspects of the chickens’ health and development. The objective of this study was to analyze and compare the composition and diversity of the microbiota in the gastrointestinal tract sections of broiler chickens. This analysis was performed by 454 pyrosequencing using the V3–V6 region of the 16S rRNA genes. Samples collected from 14 healthy, 42 d old Cobb broiler chickens revealed that the dominant bacterial phyla in the seven gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. At the genus level, Lactobacillus was predominant in the upper gut (gizzard, proventriculus, duodenum, and jejunum) and ileum, whereas unclassified Lachnospiraceae, unclassified Ruminococcaceae, unclassified Synergistaceae, Bacteroides, and Ruminococcus were predominant in the lower gut (ceca and cloaca). Eight lactic acid bacteria species were found in the gastrointestinal tract sections of chickens. Principal coordinates analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: the upper gut, ileum, and lower gut. Venn diagrams illustrated the distribution of shared and specific operational taxonomic units among the different gastrointestinal tract sections. Our results might be attributed to the probiotic development and performance improvement of broiler chickens.