This study is aimed to determine the relationship between drying, seed physiology, oxidative stress-induced lipid peroxidation and chemistry of lipid peroxidation products during drying and viability loss of Quercus glauca seeds. We used labeled phosphatidylcholines, LA-PC/D₃ 2 and DHA-PC/Et 3, as molecular probes, and analyzed them using tandem electrospray ionization mass spectrometry (ESI MS). When the seeds of Quercus glauca (Fagaceae) were slowly dried to 50.6%, 36.7% and 23.1% water content, they germinated to 90%, 62% and 2%, respectively, indicating this species produces desiccation-sensitive seeds. During drying, the amounts of 2-thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation), significantly increased in the seeds, and then decreased, in line with loss of viability. However, TBARS are terminal compounds formed from phospholipids via multiple steps, and the chemistry is complex. The experiments revealed that the extent of the oxidative decomposition of synthetic phospholipid 3 added to Q. glauca seed embryos was dependent on seed moisture content, the exposure time of the phospholipid and embryo mass. The significant correlations between the moisture content, embryo lipid peroxidation and seed viability loss indicate that desiccation stress resulted in viability loss due to cell membrane damage. These results are consistent with reactive oxygen species (ROS)-mediated lipid peroxidation determining the physiology of the drying-sensitive seeds.