During the transition from incubation to hatch, the chicks shift from obtaining nutrients from the yolk sac to the intestine. The yolk sac tissue (YST) and small intestine serve as biological barriers between the yolk or gut contents and the blood circulation. These barriers must maintain structural integrity for optimal nutrient uptake as well as protection from pathogens. The objective of this study was to investigate the effect of high incubation temperature on mRNA abundance of the tight junction (TJ) proteins zona occludens 1 (ZO1), occludin (OCLN), claudin 1 (CLDN1), and junctional adhesion molecules A and 2 (JAMA, JAM2) and the heat shock proteins (HSP70andHSP90) in the YST and small intestine of embryonic broilers. Broiler eggs were incubated at 37.5°C. On embryonic day 12 (E12), half of the eggs were switched to 39.5°C. YST samples were collected from E7 to day of hatch (DOH), while small intestinal samples were collected from E17 to DOH. The temporal expression of TJ protein mRNA from E7 to DOH at 37.5°C and the effect of incubation temperature from E13 to DOH were analyzed by one-way and two-way ANOVA, respectively and Tukey's test. Significance was set atP< 0.05. The temporal expression pattern of ZO1, OCLN, and CLDN1 mRNA showed a pattern of decreased expression from E7 to E13 followed by an increase to DOH. High incubation temperature caused an upregulation of ZO1 and JAM2 mRNA in the YST and small intestine. Using in situ hybridization, OCLN and JAMA mRNA were detected in the epithelial cells of the YST. In addition, JAMA mRNA was detected in epithelial cells of the small intestine, whereas JAM2 mRNA was detected in the vascular system of the villi and lamina propria. In conclusion, the YST expressed mRNA for TJ proteins and high incubation temperature increased ZO1 and JAM2 mRNA. This suggests that the TJ in the vasculature of the YST and intestine is affected by high incubation temperature.