Through selective culturing/adaptation we have developed a new strain of Saccharomyces cerevisiae 424A(LNH-ST) with improved xylose fermentation and ethanol resistance compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6% w/v) than the original strain. Since membrane lipid composition is expected to be one of the core factors to increased ethanol resistance, it is important to know the dynamics of the lipidome to understand molecular basis of the improved resistance. We analyzed lipids compositions in our original strain 424A(LNH-ST) and the new 424A(LNH-ST)-ER strain during xylose fermentation with and without ethanol addition. The concentrations of 16 free fatty acids (FFAs) were quantified by using QQQ LC/MS/MS mass spectrometry. Based on the results, stearic acid and palmitic acid were the predominant FFAs present in yeasts under the tested conditions. Interestingly, the saturated /unsaturated ratios of the FFAs were significantly different between the two strains. The 424A(LNH-ST)-ER strain contained higher concentrations of saturated FFAs than the original strain and the ratio of saturated/unsaturated FFAs decreased as ethanol concentration increased. The ratios and amounts of glycerophospholipids and sphingolipids in the cell membrane were also investigated. We have established a new approach for comparative and semi-quantitative lipidomics using RPLC (C8) coupled to LTQ-Orbitrap mass spectrometry. After data normalization of four replicate experiments, ∼70 lipid species were identified based on their molecular signatures, in which ∼30% of the species showed at least 2 fold change with a significance p>0.05 between the control and the enhanced ethanol resistant strain.