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Abstract The utilization of food resources by aquatic consumers reflects the structure and functioning of river food webs. In lotic water systems, where food availability and predator–prey relationships vary with gradient changes in physical conditions, understanding diet assimilation by local communities is important for ecosystem conservation. In the subtropical Liuxi River, southern China, the relative contribution of basal resources to the diet assimilation of functional feeding groups (FFGs) was determined by stable carbon (13C) and nitrogen (15N) isotope analyses. The output of Bayesian mixing models showed that diatom‐dominated periphyton (epilithic biofilm), aquatic C3 plants (submerged hydrophytes), and suspended particulate organic matter (SPOM) associated with terrestrial C3 plants contributed the most to the diet assimilation of FFGs in the upper, middle, and lower reaches, respectively. The relative contribution of consumer diet assimilation was weighted by the biomass (wet weight, g/m2) of each FFG to reflect resource utilization at the assemblage level. From the upper to the lower reaches, the spatial variation in the diet assimilation of fish and invertebrate assemblages could be summarized as a longitudinal decrease in periphyton (from 57%–76% to