Current crop and climate modeling studies predict temperature extremes that may add future challenges to global food and agriculture production systems due to yield decreases in our staple cereal crops. Although there have been some temperature stress adaptation traits and a few associated genes discovered in plants for enhanced thermotolerance, very little is known about these traits in our major cereal crops, particularly in maize. Furthermore, the limited availability of appropriate selection environments and accurate phenotyping, including functional traits for selection, have been major hurdles to overcome in making meaningful gains towards improved thermotolerance in breeding programs. Previous reports have established that dynamic changes in leaf membrane lipids occur when exposed to temperature stress and many have tried to identify specific lipid classes, individual species, or levels of unsaturation as indicators of tolerance or susceptibility. In this study, several types of cereals, with special emphasis on maize, are studied with the goal to expand the understanding of the leaf lipid membrane characteristics and responses when exposed to temperature stress and to find evidence of heritable lipid biomarker(s) that could be used in breeding for enhanced thermotolerance. Leaf lipids for maize inbred lines and twelve hybrids exhibiting differential tolerance to high temperature stress were analyzed after growing in a controlled environment at optimal and high temperature stress conditions. It was hypothesized that the newly introduced ratios of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) containing acyl chains with total 36 carbons and 6 double bonds (36:6) compared to those with 36 carbons and 5 double bonds (36:5), here termed the MGDG and DGDG “Unsaturation Ratios” can be used to differentiate the changes in the plastidic lipid membrane unsaturation levels and to aid in identifying heat tolerant genotypes. An analysis of the MGDG and DGDG Unsaturation Ratios was performed on twenty-five diverse parents of the nested-association mapping (NAM) population, Mo17, and one hundred and ninety-one B73 x B97 recombinant inbred lines (RILs) grown in field conditions. The selected lipid phenotypes were found to be as diverse as the inbred lines in which they were measured and showed a large differential between the temperate inbred lines B73 and B97. Allelic variation controlling the differences in MGDG and DGDG Unsaturation Ratios was identified in the B73 x B97 RIL subpopulation through linkage mapping analysis. Finally, an analysis of the MGDG and DGDG Unsaturation Ratios was performed across eight of the world’s most important cereal crops. The results of these studies provide preliminary evidence that the MGDG and DGDG Unsaturation Ratios may be beneficial lipid biomarkers that can be used to screen germplasm in breeding programs for improved thermotolerance for our most important cereal crops with the potential to differentiate tolerance in germplasm even without the presence of the ideal selection environment.