Structural changes in the intestinal microbial flora of laying hens were analyzed to determine the influence of heat stress on the intestinal microbial environment and explore microbial colonization of the poultry intestinal tract in the hot environment. Ninety-six 16-week-old Jining Bairi chickens were randomly allotted into control group Ⅰ (24±1)℃ or the heat stress (38±1)℃ group. Each group was divided into six replicate subgroups, each consisting of eight individuals. The duration of the experiment was 14 days. Denaturing gradient gel electrophoresis combined with multivariate statistical analysis and real-time quantitative analysis of the 16S rDNA were used to analyze the diversity of the microflora in the duodenum, jejunum, and ileum. Changes in the abundance of the microflora were also studied. The effect was examined at 2 days (group Ⅱ), 7 days (group Ⅲ), and 14 days (group Ⅳ) of heat exposure. PCA was carried out and the results showed that the composition of the microflora in parts of the duodenum remained similar between the heat-stressed and control groups, but that the microbial composition of the jejunum area varied between the heat-stressed and control animals after 2 days. At 7 days, significant differences were found in the composition of the microflora in the jejunum and ileum compared with that at 2 days and at 14 days. The influence of heat stress on the bacterial composition in each part of the intestinal tract was most pronounced at the 7-day time point. Bacilli, Lactobacillus agilis in the jejunum and ileum, and Lactobacillus johnsonii and other uncultured bacteria in the ileum, were not detected at the 7- or 14-day time points, while Bacteroidetes and γ-Proteobacteria, uncultured Escherichia sp, Bacteroides helcogenes, Bacteroides ovatus, and Shigella sonnei, were detected in the jejunum and ileum at different time points. Decreases in the overall abundance of L. johnsonii and L. agilis were most obvious in the jejunum (P<0.05), while the abundance of B. ovatus and uncultured Escherichia sp. in the jejunum and ileum was obviously increased (P<0.05). The number of bacterial cells of each of the various strains ranged between 1.76-6.58 lg CFU/g. The results indicated that the proliferation of lactobacilli and uncultured bacteria in the jejunum and ileum of laying hens was inhibited under heat stress, while the growth of B. ovatus in the jejunum and ileum was promoted, resulting in disruption of the digestive tract microflora balance. The intestinal microflora of laying hens under heat stress is relatively abundant, and the diversity of the flora in the jejunum and ileum showed distinct patterns with increasing heat stress exposure time. The effect of heat stress on jejunal microbial flora was most pronounced, followed by the ileum and the duodenum. The types of lactobacilli in the jejunum and ileum were significantly different at the different time points. The effects of thermal stress were detected in the duodenum and jejunum, particularly in reference to four kinds of pathogenic bacteria. The results suggest that heat stress can inhibit Lactobacillus species and promote the proliferation of oval Bacteroidetes, thereby disrupting the balance of the digestive tract microflora.