Heat stress (HS) has a negative effect on poultry production sustainability due to its adverse consequence on bird welfare, health, growth, and mortality. Although modern broilers have greater gut mass and higher energy use efficiency than unselected birds, they are more vulnerable to HS that induces “leaky gut syndrome,” or increased intestinal permeability. The aim of the current study was to determine the effect of HS on growth performance and gut barrier integrity in three modern broiler lines and their ancestor the Jungle Fowl. Four chicken populations including Giant Jungle Fowl (JF), Athens Canadian Random Bred (ACRB), 1995 Arkansas Random Bred (95RAN), and Modern Random Bred (MRB) were studied. Day-old male broiler chicks from each population were raised under thermoneutral (TN) conditions with feed intake, water intake, and temperature measured daily. On day 28 the birds were subjected to one of two environment conditions: TN (24°C) or acute HS (2 hrs at 36°C). After two hours, samples from each section of the small intestine were harvested from two birds per line per treatment and flash frozen in liquid nitrogen. Following 28, the remaining birds were grown out to 56, during which birds were subjected to chronic cyclic HS (8 hrs a day at 36ºC). Growth performance, metabolite and blood hormone concentrations, and molecular data were analyzed by two-way ANOVA. These data show the significant effect HS had on growth performance and intestinal barrier integrity of the studied modern broilers. Acute HS was shown to decrease performance in the modern broilers and had significant effect on mRNA and protein expression of heat shock, tight junction, gap junction, and other intestinal barrier associated proteins. These data provide evidence for a mechanistic understanding of gut barrier physiology and how it can be influenced by growth-rate and heat stress