Background and Aim: Thermal stress (hot or cold) is one of many environmental stressors that severely affects the health of broiler chickens. One negative effect of thermal stress is the disruption of the intestinal barrier function in broiler chickens. This study aimed to evaluate the effect of thermal manipulation (TM) on the small intestine in terms of histomorphometry as well as junctional, heat-shock, and immune response gene expression during post-hatch exposure to thermal stress. Materials and Methods: The experiment was conducted by dividing 928 fertile Ross eggs into three incubation groups: The control (C) group (incubated at 37.8[degrees]C and 56% relative humidity [RH] for the whole incubation period), the TM using low temperature TML group (incubated at 36[degrees]C and 56% RH for 18 h/day from embryonic days 7 to 16), and the TM using high temperature (TMH) group (incubated at 39[degrees]C and 65% RH for 18 h/day from embryonic days 7 to 16). On post-hatch day 21, 90 chicks were randomly selected from each incubation group and were equally subdivided into three subgroups for the post-hatch thermal stress experiment: The TN subgroup (room temperature maintained at 24[degrees]C), the heat stress (HS) subgroup (room temperature maintained at 35[degrees]C), and the cold stress (CS) subgroup (room temperature maintained at 16[degrees]C). After 1 day of thermal stress exposure (age 22 days), five birds from each subgroup were euthanized and ileum samples were collected to evaluate the transcription of the Claudin (CLDN1), CLDN-5, Occludin, Cadherin-1, heat shock factors (HSF1), HSF3, 70 kilodalton heat shock protein, 90 kilodalton heat shock protein, Interleukin 6 (IL6), IL8, toll-like receptors-2 (TLR2), and TLR4 genes by Real-Time Quantitative Reverse Transcription polymerase chain reaction analysis. Finally, after 4 and 7 days of thermal stress (age 25 and 28 days, respectively), nine chicks were euthanized, and their jejunum and ileum were collected for histomorphometric analysis. Results: After exposure to 1 day of thermal stress, the C subgroups exposed to thermal stress (HS and CS) possessed significantly increased expression of junctional, heat-shock, and immune response genes compared to the C-TN subgroup, and similar results were observed for the TMH. In contrast, thermally stressed TMH subgroups had significantly lower expression of the studied genes compared to C subgroups exposed to thermal stress. Furthermore, no significant changes were detected between the TML subgroups exposed to thermal stress and TML-TN. Moreover, significant alterations in villus height (VH), villus surface area, crypt depth (CD), and VH to CD ratio were observed between the TML, TMH, and C subgroups exposed to CS. Conclusion: It might be suggested that TM may have a protective impact on the small intestine histomorphometry and epithelial integrity of broilers during post-hatch exposure to thermal stress. Keywords: broilers, heat shock proteins, immune response, junctional proteins, thermal manipulation, thermal stress.