The fatty acids composition and relative content were analyzed by gas chromatography-mass spectrometry (GC-MS), which were extracted from the mycelia of Stropharia rugoso-annulata Farlow apud Murrill strains Gaoyou, Zhaoyang and Tufei. The results showed that 15 kinds of saturated fatty acids and 18 kinds of unsaturated fatty acids were found in Gaoyou and Zhaoyang mycelia. The fatty acids composition in Tufei mycelia consisted of 11 kinds of saturated fatty acids and 17 kinds of unsaturated fatty acids, which were less than those in Gaoyou and Zhaoyang mycelia. The relative content of unsaturated fatty acids was higher than that of saturated fatty acids in three stains of Stropharia rugoso-annulata, accounting for 77.4%, 77.5% and 78.7% of the total fatty acids in Gaoyou, Zhaoyang and Tufei, respectively. Linoleic acid was the main unsaturated fatty acid, which were 76.8%, 88.5% and 72.8% of the total unsaturated fatty acids in mycelia of Gaoyou, Zhaoyang and Tufei, respectively. Palmitic acid was the main saturated fatty acid, which accounted for 64.3%, 69.2% and 62.5% of the total saturated fatty acids in mycelia of Gaoyou, Zhaoyang and Tufei, respectively.