Instream barriers can constrain dispersal of nonnative fishes, creating opportunities to test their impact on native communities above and below these barriers. Deposition of sediments in a river inflow to Lake Powell, USA resulted in creation of a large waterfall prohibiting upstream movement of fishes from the reservoir allowing us to evaluate the trophic niche of fishes above and below this barrier. We expected niche overlap among native and nonnative species would increase in local assemblages downstream of the barrier where nonnative fish diversity and abundance were higher. Fishes upstream of the barrier had more distinct isotopic niches and species exhibited a wider range in δ15N relative to downstream. In the reservoir, species were more constrained in δ15N and differed more in δ13C, representing a shorter, wider food web. Differences in energetic pathways and resource availability among habitats likely contributed to differences in isotopic niches. Endangered Razorback Sucker (Xyrauchen texanus) aggregate at some reservoir inflows in the Colorado River basin, and this is where we found the highest niche overlap among species. Whether isotopic niche overlap among adult native and nonnative species has negative consequences is unclear, because data on resource availability and use are lacking; however, these observations do indicate the potential for competition. Still, the impacts of diet overlap among trophic generalists, such as Razorback Sucker, are likely low, particularly in habitats with diverse and abundant food bases such as river-reservoir inflows.