Fatty acid compositions of both the polar phospholipids (PLs) and the non-polar neutral lipids (NLs) in fish tissues are influenced by dietary fatty acids, and tissue fatty acid compositions also change during thermal acclimation. The interaction between these factors in governing fatty acid compositions has been little studied, even though this may have importance when fish are reared in cold water. An experiment was conducted to investigate the effects of temperature (2 vs. 8°C), dietary oil source (fish oil vs. vegetable oils) and feed fat content (21% vs. 34% fat) on tissue fatty acid compositions of Atlantic salmon parr. The fish were held in fresh water under a 12 h light:12 h dark photoperiod until they doubled in body mass (from ca. 19 to 38 g, which took ca. 2 months at 8°C and ca. 6 months at 2°C), and then the muscle, viscera and carcass PLs and NLs were analysed for fatty acid composition. The ratios of unsaturated to saturated fatty acids (UFA:SFA), and the unsaturation indices (UIs, the number of unsaturated bonds per 100 fatty acid molecules) were calculated for each lipid class (PL, NL) within each tissue (muscle, viscera and carcass). Both dietary fatty acids and temperature influenced the compositions of the tissue lipids, with the dietary effects being most pronounced. The fatty acid composition of the feed oils was strongly reflected in the NLs of all three tissues, and also had a significant influence on the fatty acid profiles of the PLs. The effects of temperature were more marked in the PLs than in the NLs. Exposure to the lower temperature resulted in PLs with higher UFA:SFAs, and this is interpreted as a thermal acclimation response that would contribute to the maintenance of membrane fluidity. The PLs of fish given vegetable oils had higher UFA:SFAs than those of salmon provided with feeds containing fish oil, implying that the cell membranes of the former may have had greater fluidity. By contrast, the PLs of salmon fed with fish oil had higher concentrations of n-3 highly unsaturated fatty acids, and higher UIs, which may have made them more prone to peroxidative damage. This raises the intriguing possibility that the feeding of vegetable oils may have produced fish that were better able to withstand exposure to low temperature as a result of improved membrane fluidity (implied from the higher UFA:SFAs in these fish), while having membrane PLs that were less susceptible to oxidative damage (implied from the lower UIs).