Although several studies have analyzed the fatty acid profile of phospholipids (PL) and, to a lesser degree, triacylglycerols (TG) in one or more tissues concurrently, a systematic comparison of the fatty acid composition of different tissues and/or lipid classes is lacking. The purpose of the present study was to compare the fatty acid composition of major lipid classes (PL and TG) in the rat serum, soleus muscle, extensor digitorum longus muscle and the heart. Lipids were extracted from these tissues and analyzed by a combination of thin-layer chromatography and gas chromatography. We found many significant differences in various tissues and lipid classes. Serum had the most distinct fatty acid profile in PL but this “uniqueness” was less apparent in TG, where differences among tissues were in general less frequent than in PL. These two skeletal muscles exhibited similar fatty acid composition in both lipid classes despite their different muscle fiber type composition, denoting that fiber type is not a major determinant of the fatty acid composition of rat skeletal muscle. The fatty acid profile of heart PL was the most different from that of the other tissues examined. PL were rich in polyunsaturated fatty acids, whereas TG were rich in monounsaturated fatty acids. Although the reasons for the differences in fatty acid profile among the tissues examined are largely unknown, it is likely that these differences have an impact upon numerous biological functions.