The fatty acid composition of muscle membrane phospholipids and fat stores may affect migration performance in birds. The purpose of this study was to investigate seasonal changes in the fatty acid composition of (1) pectoralis muscle phospholipids, (2) intramuscular triglyceride stores and (3) adipose tissue triglycerides in free-living white-throated sparrows (Zonotrichia albicollis). During migratory seasons there was an increase in the n-6:n-3 ratio of muscle membrane phospholipid fatty acids without a change in the proportion of unsaturated fatty acids. This change was driven mainly by an increase in the proportion of 18:2n-6 and a decrease in the proportion of 22:6n-3. An increase in the proportion of 18:2n-6 was also observed in the intramuscular and adipose tissue triglyceride stores during the migratory seasons. These increases in 18:2n-6 were offset by a decrease in 16:0; resulting in an elevated proportion of unsaturated fatty acids and elevated double bond index in both fat stores of migrants. The elevated levels of 18:2n-6 in migrant fat stores indicates a high dietary component of this fatty acid, as white-throated sparrows feed mainly on tree seeds and some insects during migration and may not have access to a diet high in n-3 fatty acids. We suspect that elevated dietary levels of 18:2n-6 also caused the observed increases in the proportion of this fatty acid in muscle phospholipids. Overall, we conclude that seasonal changes in adipose and muscle fatty acid composition are likely attributable to diet more than other factors such as migratory exercise or mitochondrial density.