Understanding the changes in peanut (Arachis hypogaea L.) anther lipidome under heat stress (HT) will aid in understanding the mechanisms of heat tolerance. We profiled the anther lipidome of seven genotypes exposed to ambient temperature (AT) or HT during flowering. Under AT and HT, the lipidome was dominated by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG) species (> 50% of total lipids). Of 89 lipid analytes specified by total acyl carbons:total carbon-carbon double bonds, 36:6, 36:5, and 34:3 PC and 34:3 PE (all contain 18:3 fatty acid and decreased under HT) were the most important lipids that differentiated HT from AT. Heat stress caused decreases in unsaturation indices of membrane lipids, primarily due to decreases in highly-unsaturated lipid species that contained 18:3 fatty acids. In parallel, the expression of Fatty Acid Desaturase 3-2 (FAD3-2; converts 18:2 fatty acids to 18:3) decreased under HT for the heat-tolerant genotype SPT 06-07 but not for the susceptible genotype Bailey. Our results suggested that decreasing lipid unsaturation levels by lowering 18:3 fatty-acid amount through reducing FAD3 expression is likely an acclimation mechanism to heat stress in peanut. Thus, genotypes that are more efficient in doing so will be relatively more tolerant to HT.