Fatty acid (FA) analyses of fish tissues offer the potential to gain new knowledge of habitat or forage-specific energy inputs to fishes in river-floodplain ecosystems, although limited information exists regarding among-habitat differences in FA biomarkers. The goal of this study was to determine if differences in fish FA profiles among main channel and connected and disconnected floodplain lakes exist in large river-floodplain systems. Bluegill Lepomis macrochirus FA profiles were generated to assess differences among two reaches of the Illinois River, USA, and its connected and disconnected floodplain lakes and determine whether FA signatures could be used to reclassify fish to their source habitat. Bluegill FA profiles differed among habitats and river reaches, including differences in levels of individual FAs (e.g., 18:2n-6, an indicator of allochthonous inputs, was higher among main channel fish) and FA groupings (e.g., n-3:n-6 FA ratio, an indicator of aquatic primary productivity, was higher among floodplain lake fish), which enabled [87.5% reclassification accuracy of fish to their source environment. We demonstrated that bluegill FA profiles differed among reaches and laterally among river channel and floodplain habitats, suggesting that FA profiles can be used to infer recent habitat use and habitat-specific foraging of fishes in large river-floodplain ecosystems.