Abstract: The composition and content of fatty acids in edible cassava flour not only affect its shelf life but also serve as important indicators for evaluating the nutritional quality of cassava flour. In this experiment, edible cassava flour was used as the sample. By optimizing the fatty acid methyl esterification method and gas chromatography conditions, a qualitative and quantitative detection method for fatty acids in edible cassava flour was established. The fatty acid composition in edible cassava flour of different varieties (strains) and different storage times was analyzed and detected. The results showed that gas chromatography could accurately analyze and quantify various fatty acids in edible cassava flour. There was a good linear relationship between the concentrations of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid in the ranges of 20.0~1000.0, 4.0~400.0, 30.0~1500.0, 10.0~500.0 and 2.0~100.0 μg/mL, respectively, with coefficient of determination (R²) ranging from 0.9992 to 0.9999. The relative standard deviations (RSDs) of sample repeatability ranged from 0.5% to 3.2%. The RSDs of concentration changes after 24 hours of storage at room temperature ranged from 0.7% to 1.1%. The average recovery rates of the five fatty acids in the samples ranged from 88.0% to 105.4%, with average recovery rate RSD ranging from 3.4% to 10.4%, indicating good sample stability, method repeatability, and accuracy. The cassava flour prepared from different edible cassava varieties mainly contained palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. There were significant differences in fatty acid content among different varieties, and the proportions of the five cassava flour fatty acids (SFA/MUFA/PUFA) were 1:(0.91~1.35):(0.12~0.39). Among them, the content of palmitic acid, oleic acid, linoleic acid, and linolenic acid in 'Huanan 9' cassava flour was significantly higher than other varieties (P<0.05). The content reached 0.7818, 1.088, 0.2967 and 0.0398 mg/g, respectively. However, the content of stearic acid in this variety was the lowest, only 0.0868 mg/g. Ambient temperature storage experiments showed a negative correlation between the content of the five fatty acids and storage time, with oleic acid, linoleic acid, and linolenic acid showing a highly significant negative correlation (P<0.01). The content of five fatty acids reached its lowest level after 12 weeks of storage, and the changes tended to be gradual in the later storage stages. The research results provide a basis for evaluating the composition of fatty acids in edible cassava flour of different varieties (strains) and the changes in fatty acids during the storage process.