Increasing prevalence of hypoxia in shallow waters of U.S. Gulf of Mexico (GoM) estuaries can pose a serious threat to eastern oysters (Crassostrea virginica). Their tolerance to hypoxia, however, is not well characterized, especially at elevated temperatures (>30 °C) typical of GoM estuaries in summer. Moreover, it is unknown whether differences in hypoxia tolerance exist between GoM oyster populations growing in estuaries differing in local environmental conditions. Wild oyster broodstocks were collected from four estuarine sites in Texas (Packery Channel, PC and Aransas Bay, AB) and Louisiana (Calcasieu Lake, CL and Vermilion Bay, VB) and their adult progenies (F1) were tested (Study 1) under continuous hypoxia (<2.0 mg O₂ L⁻¹) at 32 °C. Significant differences in hypoxia tolerance were found between F1 populations with calculated median lethal time (LT₅₀) ranging from 3.9 to 12.5 days. PC and CL oysters were the most and least tolerant populations, respectively. The study was repeated twice more (Studies 2 and 3) using PC and CL oysters, and their responses at the organismic, cellular, and biochemical levels were investigated. Valve movement was monitored, and oysters were sampled to measure hemocyte density, plasma protein, calcium and glutathione concentrations, and digestive gland alanine and succinate concentrations after either 3–5 days (Study 2) or 1–3 days (Study 3) of hypoxia exposure. From the onset of hypoxia until their death, oysters stayed opened 13–32% of the time compared to 53–64% under normoxia, but no differences between populations were detected under hypoxia. PC oyster but not CL oyster plasma glutathione concentrations increased significantly in both studies. Under longer (3–5 days) hypoxia exposure, plasma calcium and glutathione concentrations of PC oysters were significantly higher than CL oysters. These results suggest PC oysters were better able to protect tissues against acidosis and oxidative damage during hypoxia and high temperature stress than CL oysters. Overall, our results indicate that oyster populations originating from the GoM vary in their response to hypoxia and high temperature stress and possess differential tolerance.