© 2021 John Wiley & Sons LtdThe present study evaluated the effects of dietary fish oil replacement with linseed or sesame oil on sperm quality and reproductive performance in rainbow trout (Oncorhynchus mykiss) during a 7-month feeding trial. Male (≈ 970 g) broodfish were fed three isonitrogeneous and isolipidic formulated diets containing fish oil (FO diet), linseed oil (LO diet) and sesame oil (SO diet). Commercial trout broodfish diet was fed to another group to serve as control (CD). Growth performance was not affected by diet. Slight variations in volume, pH and density were recorded in the semen produced by rainbow trout subjected to the different dietary treatments (p > 0.05). Similarly, motility and kinematic parameters were not influenced by diets (p > 0.05). However, osmolality differed in the dietary groups, with semen from SO and LO fed fish having comparatively higher values (p < 0.05). Fatty acid (FA) profile of fish spermatozoa reflected dietary FA composition. FA profile of spermatozoa showed evidence of de novo biosynthesis of eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid. Dietary treatments did not significantly affect reproductive performance (fertilization, eyeing and hatching rates). This may be suggestive of the ability of male rainbow trout to synthesize HUFA in vivo to meet their reproductive needs in the event of low dietary n-3 HUFA supplementation.