Objective To investigate the effects of different sample pretreatment methods on detection of fatty acids in fruit drugs of Rosaceae by gas chromatography-mass spectrometry (GC-MS), and to provide an experimental basis for sample pretreatment conditions. Methods Direct methyl esterification (DME) and ethanol extracts methyl esterification (EEME) were used for pretreatment of the power of crude drugs (Chaenomeles sinensis, Fructus Mume, Crataegus pinnatifida, Rubus idaeus, and Rosa laevigata) from the Rosaceae family by H2SO4-CH3OH (1∶10) solution. And then GC-MS was used to detect fatty acids in samples, and the results were compared between the two pretreatment methods. Results DME identified more fatty acids (C2 to C24); the number of fatty acids identified by EEME changed with the polarity of extraction solvent: the higher polarity was beneficial to the detection of short-chain fatty acids such as malic acid and critic acid, while the lower polarity was beneficial to the detection of long-chain fatty acids such as oleic acid and linoleic acid. Conclusion Different sample pretreatment methods lead to difference in the detection of fatty acids by GC-MS. Under the same analytic conditions, DME can be used to understand the types of fatty acids in a comprehensive way; the types of fatty acids identified by EEME are less and related to the polarity of the extraction solvent.