The lipid and fatty acid compositions of the total lipids of three cultured populations (migratory between fresh and salt water, Lake Biwa landlocked, and Setogawa River forms) of ayu, Plecoglossus altivelis, were investigated to clarify the difference in lipid characteristics and temperature adaptability among the three groups. Triacylglycerols were the dominant depot lipids of the three populations, while phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, were found to be the major components of the polar lipids, and their lipid classes are similar to each other. The major fatty acids in the triacylglycerols of all specimens were 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, 18:2n-6 (linoleic acid), 20:5n-3 (EPA, icosapentaenoic acid), and 22:6n-3 (DHA, docosahexaenoic acid), similar to the tissue phospholipids of the three populations, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, 18:2n-6, 20:4n-6, EPA, and DHA. All classes had high levels of 18:2n-6, which originates from their dietary lipids. Compared with the lower DHA levels of the triacylglycerols, the higher levels in the phospholipids suggest their selective accumulation or a biosynthetic pathway to DHA as in freshwater fish. Two populations (the migratory and Setogawa River forms) adapted to lower temperatures with comparatively high levels of polyunsaturated fatty acids (PUFA) for their membrane fluidities. With significantly higher levels of n-3 PUFA and total PUFA, the mean DHA content in the lipids of the Setogawa River form (the population that adapted to lower temperatures) was significantly higher than that of the migratory form. From these results, we concluded that the Setogawa River population actively concentrates long-chain PUFA in its polar lipids and has high adaptability to low temperature.