The purpose of this study was to develop a gas chromatographic (GC) method to analyze fatty acids in milk fat with a single injection. The single-injection GC method we developed for analyzing fatty acid composition can separate a wide range of fatty acid methyl esters from butyric acid to docosahexaenoic acid. It separated 6 isomers of 18:1 (cis-6, cis-9, cis-11, trans-6, trans-9 and trans-11), 4 isomers of 18:2 (cis-9-cis-12, trans-9-trans-12, cis-9-trans-12 and trans-9-cis-12), and 4 isomers of conjugated 18:2 (cis-9- trans-11, trans-9-cis-11, cis-10-trans-12 and trans-10-cis-12).