Triploid eastern oysters (Crassostrea virginica) suffer greater mortalities than diploids in the U.S. Gulf of Mexico estuaries when extreme low salinities (< 5) and elevated temperatures (≥ 28 °C) coincide. To investigate potential causes, changes in plasma osmolality, hemolymph pH, valve opening and mortality in diploid and triploid oyster half-siblings were compared during a step-down gradual acclimation from a salinity of 5 to 1 (5, 2.5, 2.0, 1.5, 1.0) at 23 °C (expt 1) and at 28 °C (expt 2). To further explore differences in diploid and triploid oyster responses to changing salinity, we compared their plasma osmolality after abrupt decreases in salinity from 20 to 10 and 5, followed by increases in salinity from 5 and 10 to 20 once oysters had osmoconformed to the lowered salinities (expt 3). Lastly, changes in wet weights of mantle tissue explants were compared between diploid and triploid oysters every 10 min for 40 min after being transferred from a salinity of 20 to 10 (expt 4). Oysters of both ploidies were able to osmoconform to water at salinities between 5 and 1.5. After a decrease in salinity, triploid oysters were slower to open their valves and osmoconform, were less efficient in maintaining acid-base status enduring longer periods of acidic hemolymph pH, and were less efficient in regulating tissue water content compared to half-sibling diploid oysters. At a salinity of 1.0, plasma of both diploid and triploid oysters remained hyperosmotic, their hemolymph acidic and their valves closed. Oysters osmoconformed faster at 28 °C than at 23 °C, but the combination of low salinity (≤ 1.5) and higher temperature caused rapid mortalities regardless of ploidies. Triploid oysters, however, started dying earlier and at greater percentages when salinity of 1.5 and temperature of 28 °C were combined. Triploids have been embraced as a means to support higher production, but results indicate superimposed stressors, such as low salinity and high temperature, may be more lethal to triploid than diploid oysters.