OBJECTIVE： To establish a method for simultaneous determination of fatty acids in Isaria cicadae， Cordyceps militaris and C. sinensis， and to compare the difference of the contents of fatty acids among above medicinal herbs. METHODS： GC-MS method was adopted. Chromatographic condition： the determination was performed on TG-5MS gas phase capillary column with carrier gas of nitrogen at the flow rate of 1.2 mL/min. The inlet temperature was 290 ℃ by splitless sampling； valve opening time was 1 min， and volume of sample was 1 μL. Mass spectrometry condition：the ion source is an electrospray ionization source. The temperatures of ion source and transmission line were 280 ℃， the initial temperature of the chromatographic column was 80 ℃（gradient elution）， ionization voltage was 70 eV. Solvent delay time was 5 min， and scan mass range was m/z 30-400. RESULTS： The linear ranges of tetradecanoic acid， pentadecanoic acid， palmitic acid， palmitoleic acid， heptadecanoic acid， heptadecenoic acid， docosahexaenoic acid， methyl oleate， linoleic acid， arachidonic acid， eicosenoic acid， diolefinic acid， eicosatrienoic acid， heneicosanoic acid， behenic acid， tricosanoic acid， lignoceric acid were 1.400-44.520 μg/mL（r＝0.999 8），2.091-93.721 μg/mL（r＝0.999 7），3.146-85.856 μg/mL（r＝0.998 2），1.664-61.444 μg/mL（r＝0.998 7），1.773-64.983 μg/mL（r＝0.999 5）， 1.781-68.421 μg/mL（r＝0.999 7），1.706-55.606 μg/mL（r＝0.999 8），1.439-47.989 μg/mL（r＝0.999 6），1.738-66.908 μg/mL（r＝0.999 6）， 2.086-94.206 μg/mL（r＝0.999 5），1.356-44.966 μg/mL（r＝0.999 4），1.444-56.814 μg/mL（r＝0.999 7），1.375-52.335 μg/mL（r＝0.999 8）， 1.512-60.312 μg/mL（r＝0.999 5），1.450-59.760 μg/mL（r＝0.999 7），1.427-58.757 μg/mL（r＝0.999 1），1.269-58.109 μg/mL（r＝0.999 3）， respectively. The limit of quantitation was no more than 1 764.71 μg/mL， and the limit of detection was no more than 529.42 μg/mL. RSDs of precision， stability and reproducibility tests were all lower than 2.0％. The recoveries were 84.87％-108.93％（RSD ranged 0.19％-2.23％，n＝6）. There were 17 fatty acids in C. militaris， 16 fatty acids in I. cicadae and 16 fatty acids in C. sinensis. The contents of unsaturated fatty acids in above medicinal herbs were higher than that of saturated fatty acids. The content of fatty acids in artificial cultivated I. cicadae was mostly higher than other medicinal herbs. COCLUSIONS： The method is simple， accurate， stable and reproducible. It can be used for simultaneous determination of fatty acids in I. cicadae， C. militaris and C. sinensis. Above 3 kinds of medicinal materials. From the perspective of fatty acid content，the quality of artificial cultivated I. cicadae is best.