Objective：To compare the differences of fatty acid composition and content in different brain regions of rats，and to provide experimental evidence for clarifying the distribution of fatty acids in the brain. Methods：Lipids were extracted from the brain regions with methanol/dichloromethane，and fatty acids were methylesterified using methanol/acetyl chloride. Fatty acid methyl esters were then assayed by gas chromatography. Results：Five brain regions including cerebellar vermis，striatum，hippocampus，cerebral cortex and white matter，contained at least 31 fatty acids. There were significant differences in the contents of the ten kinds of fatty acids（including C20∶0，C22∶0，C23∶0，C24∶0，C20∶1，C22∶1，C24∶1，C20∶5n-3，C22∶5n-6 and C22∶6n-3）among the five brain regions. Conclusion：The fatty acid compositions in the five brain regions of rats are basically the same and some fatty acid contents are significantly different.