Spatial patterns of resource use by small-bodied fishes in the San Juan River were examined using stable isotopes. Using δ¹⁵N of fishes as an index of trophic position, our data suggest both native and non-native fishes primarily consumed macro-invertebrates. The δ¹³C of these fishes further suggested a detritus-based food web, from which most species fed on chironomids in low-velocity habitats. A two-way ANOVA revealed a significant interaction between trophic level of fish species and longitudinal position in the river. This interaction was primarily attributed to a decline in trophic level of non-native red shiner Cyprinella lutrensis, relative to other species, in upstream reaches of the river. In addition, ANCOVA results suggest trophic position of fishes was dependent on channel type (primary vs. secondary), as there was less variability in resource use in secondary channels. These data provided a spatial framework of trophic interactions that can be used to predict the outcome of management actions. Overall, we confirmed high overlap in resource used between native and non-native fishes. However, spatial variation in trophic interactions both longitudinally and laterally in the river present a challenge to resource managers attempting to managing entire river systems.