Differences in reproductive strategies of male and female fishes are presumably accompanied by differences in nutrient allocation and predicted to lead to divergence in body composition between the sexes. We compared patterns of variation in fatty acid profiles of lipids extracted from ova, liver, muscle and visceral fat between mature male and female walleye (Sander vitreus) sampled from two wild spawning stocks. Fatty acid profiles differed significantly among body tissues in both males and females, with the strongest contrast between muscle and visceral fat. Significant differences in fatty acid composition between the sexes were found in liver, muscle and visceral fat tissues. Variation among sexes and populations was greater in liver than in the other tissues. Female livers had lower relative abundances of palmitic acid (PA, 16:0) and oleic acid (OA, 18:1(n‐9)), and higher relative abundances of arachidonic acid (ARA, 20:4(n‐6)), eicosapentaenoic acid (EPA, 20:5(n‐3)) and docosahexaenoic acid (DHA, 22:6(n‐3)) compared to male livers. In addition, female muscle had higher relative abundance of OA and lower relative abundance of DHA compared to male muscle. Our results illustrate the differential effects of reproductive demands on the biochemical composition of males and females and have implications for the analysis of fatty acid profiles in studies of wild fish populations.