The physiological processes affecting root physiology and growth at high soil temperatures are not well understood. Processes involved in root tolerance to high soil temperature and root lipid metabolism are much less studied than the equivalent processes in shoots, despite their importance to whole-plant response to high temperature stress and thermotolerance. The objectives of the current study were to examine the effects of high soil temperature on root lipid metabolism and compare lipid profile and molecular species of roots associated with plant tolerance to high soil temperature. Roots of two Agrostis species: thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park, and A. stolonifera (cv. Penncross) a heat-sensitive grass widely used as turf and forage grass in cool climate regions were exposed to soil temperature of either 37oC (high soil temperature) or 20oC (control) and both electrolyte leakage and lipid metabolism were examined. Root electrolyte leakage was higher at 37oC than at 20oC. The leakage in A. stolonifera at 37oC was significantly higher than in A. scabra. Total lipid content decreased at 37oC in both species; however, lipid content was higher in A. scabra. The lipids with the highest proportion were DGDG, PC and PE at both 20 and 37oC in both species. The proportion of saturated lipid molecular species was higher in A. scabra and increased at 37oC in both species. Our results suggest that root thermo-tolerance in A. scabra was associated with maintaining higher lipid con tent, and proportion of unsaturated lipid molecular species that contribute to increased membrane stability at high soil temperature in comparison with A. stolonifera; lipid metabolism may play an important role in root adaptation to high soil temperature.