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Biological invasions are expected to alter food web structure, but there are limited empirical data directly comparing invaded versus uninvaded food webs, particularly in species‐rich, tropical systems. We characterize for the first time the food web of Lake Gatun—a diverse and highly invaded tropical freshwater lake within the Panama Canal. We used stable isotope analysis to reconstruct the trophic structure of the fish community of Lake Gatun and to compare it to that of a minimally invaded reference lake, Lake Bayano. We found significant differences between the trophic structures of these two Neotropical lakes, notably that Lake Gatun's fish community was characterized by a longer food chain, greater isotopic diversity, a broader range of trophic positions and body sizes, and shifts in the isotopic positions of several native taxa relative to Lake Bayano. The degree of isotopic overlap between native and non …