Increasing hydropower expansion in hyper-diverse tropical river basins is currently threatening aquatic biodiversity on an unprecedented scale. Among the largest and most controversial of these projects is the Belo Monte Hydroelectric Complex being constructed on the Xingu River, a major Amazon tributary in Brazil. Despite the potentially large impacts, almost no baseline ecological data are available for the river’s diverse ichthyofauna. This study uses ecomorphology and stable isotope analysis to explore the functional and trophic relationships among four of the dominant families within the Xingu River rapids (Loricariidae, Cichlidae, Anostomidae, and Serrasalmidae). Morphological analysis revealed clear separation of these families based on functional traits associated with microhabitat use and foraging strategies, with the Loricariidae and Cichlidae displaying greatest functional diversity. The four families analyzed were not clearly differentiated in isotopic space defined by δ¹³C and δ¹⁵N values. Considerable overlap was observed among isotopic niches and all four families primarily assimilated material originating from phytomicrobenthos (assumed to be mainly benthic algae). Differences between morphological and trophic diversity within families provide insight into how this diverse assemblage may be partitioning niche space, which in turn has implications for population responses to hydrologic alteration.