Large river systems are often thought to contain a mosaic of patches with different habitat characteristics driven by differences in flow and mixing environments. Off-channel habitats (e.g., backwater areas, secondary channels) can become semi-isolated from main-channel water inputs, leading to the development of distinct biogeochemical environments. Observations of adult bluegill (Lepomis macrochirus) in the main channel of the Mississippi River led to speculation that the main channel offered superior food resources relative to off-channel areas. One important aspect of food quality is the quantity and composition of polyunsaturated fatty acids (PUFA). We sampled consumers from main-channel and backwater habitats to determine whether they differed in PUFA content. Main-channel individuals for relatively immobile species (young-of-year bluegill, zebra mussels [Dreissena polymorpha], and plain pocketbook mussels [Lampsilis cardium]) had significantly greater PUFA content than off-channel individuals. No difference in PUFA was observed for the more mobile gizzard shad (Dorsoma cepedianum), which may move between main-channel and off-channel habitats even at early life-history stages. As off-channel habitats become isolated from main-channel waters, flow and water column nitrogen decrease, potentially improving conditions for nitrogen-fixing cyanobacteria and vascular plants that, in turn, have low PUFA content. We conclude that main-channel food webs of the upper Mississippi River provide higher quality food resources for some riverine consumers as compared to food webs in off-channel habitats.