Recent work indicates that the establishment of bigheaded carps (Hypophthalmichthys spp.) in the United States has led to a reduction in condition of native planktivores and may detrimentally affect other trophic levels by altering the base of aquatic food webs. We used fatty acids to evaluate potential effects of bigheaded carps on taxa from multiple trophic levels in the Upper Mississippi, Illinois, and St. Croix rivers. Seston fatty acid concentrations were highest in the Illinois River lotic sites and connected backwaters and were positively associated with omega-3 highly unsaturated fatty acids, indicating that these locations had abundant, high-quality basal food resources despite hosting the greatest bigheaded carp densities. Fatty acid profiles of threeridge freshwater mussels tracked the fatty acid values in the seston and were not influenced by bigheaded carp abundances. Hydropsychid caddisflies and bluegill did not differ significantly in total fatty acids or percent lipid among spatial locations, indicating that omnivorous species may be relatively unaffected by bigheaded carps. Gizzard shad, however, exhibited the lowest fatty acid concentrations in the locations with the highest relative bigheaded carp densities, and multivariate models identified bigheaded carp densities as the predictive factor that explained the greatest amount of variability. Zooplankton abundance has been greatly reduced after bigheaded carps establishment in the Illinois River, which may explain the disconnect between the gizzard shad fatty acids and the plentiful, high-quality phytoplankton in that river. Our data provide additional evidence that bigheaded carps are negatively affecting native planktivores such as gizzard shad.