A long-term feeding trial was implemented on rainbow trout (Oncorhynchus mykiss) to assess the effects of seven alternative oils on fish performance and fatty acid metabolism. The tested oils were as follows: monola (a high oleic acid canola cultivar; MO), canola (rapeseed; CO), poultry by-product (chicken fat; PbPO), palm (PO), sunflower (SFO), high oleic acid sunflower (HOSFO) and soybean (SBO). All tested oils were included at a 75% substitution level of fish oil (FO) and were compared with a control diet containing 100% FO. PO, and to a lesser extent PbPO, exhibited impaired performance and lower digestibility values. All treatments containing low levels of saturated fatty acids (namely MO, CO, SFO, HOSFO and SBO) recorded an apparent in vivo fatty acid de novo production. The apparent in vivo fatty acid β-oxidation was proportional to fatty acid dietary supply and limited apparent in vivo fatty acid bioconversion (elongation and desaturation) was recorded, primarily acting on n-6 PUFA. In all treatments, dietary 20:5n-3 was actively bioconverted into 22:6n-3. It was shown that when some FO is provided with the diet, the in vivo fatty acid metabolism plays a minor role in determining final fatty acid make-up of fish whole bodies.