In large freshwater systems, the dominant production pathways supporting food webs are often spatiotemporally variable. We used stable isotope analysis and analysis of covariance (ANCOVA) models to investigate spatial and interannual variation in the dominant production pathways supporting fish consumers within the central basin of Lake Erie. We examined C and N stable isotope ratios of zooplankton, benthic invertebrates, and four species of fish common to nearshore areas of the central basin (yellow perch, Perca flavescens; white perch, Morone americana; rainbow smelt, Osmerus mordax; and round goby, Neogobius melanostomus) using tissue samples collected in 2017 and 2019. δ ¹³C values varied by location consistent with expected baseline differences in nutrient loading (¹³C was more enriched in the southern region) in two of six ANCOVA models. Furthermore, δ ¹⁵N values varied with individual fish size and by location in a manner consistent with spatial patterns of nutrient loading from surrounding agricultural landscapes (¹⁵N was more enriched in the northern region) and a longitudinal gradient of eutrophication, decreasing from west to east. These patterns were not exhibited by all species and did not necessarily persist across years, suggesting that additional factors (e.g., regional diet differences, river plume dynamics) also contributed to observed δ ¹³C and δ ¹⁵N variation. We suggest that spatiotemporal variation of stable isotope ratios should be accounted for in studies of trophic basis of production and food web structure in Lake Erie.