Abstract: Objective: To investigate lipid changes during the storage of barley flour, the oxidative patterns of lipids and lipase activity, and their interactions, thereby elucidating the reaction mechanisms underlying oxidative deterioration. Methods: Oxidative stability was evaluated by measuring fatty acid content, lipase activity, and malondialdehyde (MDA) value in barley flour stored for 28 days under high-temperature/high-humidity (HH), normal-temperature/normal-humidity (NN), and low-temperature/low-humidity (LL) conditions. Lipid composition changes were monitored using ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS/MS) to monitor lipid composition changes. Results: Under LL and NN conditions, changes in fatty acid value, lipase activity, and malondialdehyde value were relatively slow; After 28 days of storage under HH conditions, the fatty acid value increased from 91.08±1.33 mg/100 g to 136.26±2.62 mg/100 g, lipase activity rose from 41.04±0.58 mg/g to 47.91±0.61 mg/g, and malondialdehyde value increased from 45.41±2.10 nmol/g to 79.97±1.37 nmol/g. Lipidomics analysis identified 234 lipid molecules across 23 subclasses, including 176 significantly altered lipids. Correlation analysis revealed positive correlations between lipase activity and sterol lipids (STs), fatty acids (FAs), and glycerolipids (GLs). Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) indicated that barley lipids under HH conditions underwent significant changes with prolonged storage. Among these, triglycerides (TG) and diglycerides (DG) exhibited the highest susceptibility to oxidation and could serve as biomarkers distinguishing fresh barley from oxidized barley. Conclusion: Significant differences exist in barley flour lipids before and after storage. Enhancing storage stability by reducing lipase activity provides a theoretical basis for establishing barley shelf-life standards.