Abstract: This study aimed to investigate the influence of storage temperature on lipid oxidation in barley. Using barley stored at −24 °C as a control, treatment groups were established at 5, 15, 25, and 35 °C. Non-targeted metabolomics technology was employed to systematically analyze lipid changes during storage. The results indicated that fatty acid values and malondialdehyde (MDA) content increased significantly with rising storage temperature. Specifically, at 35 °C, these values increased by 40.40% and 38.82%, respectively, compared to those at 25 °C. Lipase and lipoxygenase activities peaked at 25 °C (533.95 U/g and 7400.00 U/g, respectively), whereas catalase and superoxide dismutase activities continuously decreased, leading to a significant reduction in barley antioxidant capacity. Lipidomic analysis revealed that high-temperature (35 ℃) storage activated glycerolipid metabolism and promoted the breakdown of neutral lipids, such as triglycerides, thereby producing substantial aldehyde and ketone oxidation derivatives. Volatile compound analysis further confirmed that the accumulation of characteristic lipid oxidation products, including undecanal, nonanal, 2-decen-1-ol, cyclohexanone, trimethylbenzene, naphthalene, and dodecane, contributed significantly to the development of off-flavors during barley storage. In conclusion, storage temperature modulates the activities of key enzymes and lipid metabolic pathways, elucidating the intrinsic mechanism by which temperature affects barley quality during storage. These findings provide a theoretical foundation for precisely controlling barley storage quality.