In this study, the changes of the fatty acid composition of phospholipid in different tissues (muscle, heart, brain and spleen) of steelhead trout ( Oncorhynchus mykiss) were analyzed when the water temperature decreased gradually from 16˚C to 12˚C, 8˚C, 6˚C, 4˚C, 2˚C and 1˚C. Three fish individuals each tank (average weight 70.32 g ± 9.12 g) were collected and used to analysis at each designed temperatures. At normal temperature (16˚C), the fatty acid composition of phospholipid of muscle and heart was similar each other. The highest concentration of saturate fatty acids (SFA) was found in the phospholipid of spleen. The brain phospholipid contained higher oleic acid (18:1n9) than the phospholipid of other tissues at 16˚C. When the environmental temperature decreased, the concentration of unsaturated fatty acids of phospholipids in all tissues increased, and accordingly the ratio pf the unsaturated to saturated fatty acids (U/S) and unsaturation index (UI) increased, indicating that steelhead trout can compensate temperature- dependent changes in membrane fluidity by remodeling the fatty acid composition of phospholipids. The changes in the fatty acid composition of phospholipid were tissue-specific. At the early stages of the experiment (16˚C to 8˚C), the fatty acid composition of phospholipid changed remarkably in muscle, heart, and spleen. When temperature decreased to less than 8˚C, an obvious response of phospholipid fatty acid was observed in all tissues. The change of phospholipid composition of steelhead trout tissues may be affected by both cold stress and starvation when the temperature decreased to 2˚C, and the change of phospholipid composition of muscle was very obvious.