Trophic structuring of complex food webs may vary at multiple spatial and temporal scales, both in terms of direct trophic connections and underlying energy pathways that support production. In large freshwater systems, the prey and primary producers that support individual higher-order consumers may vary across seasons and habitats due to differences in food availability, predator consumption patterns, seasonal succession of organisms at lower trophic levels, and heterogeneous nutrient inputs. We examined spatial and temporal variation in stomach contents, fatty acids, and stable isotopes of yellow perch (Perca flavescens) across seasons and across sites spanning approximately 200 km in Lake Erie's Central Basin (LECB). Stomach contents provided a short-term index of trophic patterns, while biochemical markers (fatty acids and stable isotopes) provided a more temporally integrated description of underlying energy pathways and trophic links. We found limited spatial variation of biochemical indicators and documented seasonal variation for all three trophic indicators, especially fatty acid profiles. Differences in stomach contents were driven by relative chironomid consumption, the most abundant prey resource, while fatty acid profiles were predominantly influenced by seasonal fluctuations in C22:6n-3 (DHA). Seasonal trends were evident in δ¹³C and δ¹⁵N; however, they varied within a narrow range of values. Our findings suggest that adult yellow perch in LECB showed little differentiation in resource use across space in 2014, but their diets and biochemical compositions varied seasonally.