Prairie streams and rivers are highly dynamic environments whose ecological processes are shaped by their hydrology. Seasonal flood and drought cycles create stark differences in habitat and resource quality and availability. As river channels expand and contract, the extent to which fishes exploit allochthonous and autochthonous resources is poorly described for many species. Understanding the links between seasonal hydrologic conditions, resource partitioning, and influences on age, growth, and productivity is needed to protect the viability of endemic and sensitive fish populations, especially in semi-arid regions. Here, we examined spatiotemporal variation in the upper Red River food web and characterized resource use and overlap among four sensitive prairie fishes (plains minnow; prairie chub; Red River shiner, Red River pupfish) using stable isotopes (δ15N, δ13C) and gut content analysis. Detritus, benthic algae, aquatic and terrestrial invertebrates, and fishes were sampled for stable isotopes during spring, summer, and fall of 2023. Food web structure, basal resource use, and fish diet composition varied across seasons and longitudinal gradients. For most fish, reduced δ15N and δ13C values were observed from upriver to downriver. Trends in δ13C also tracked depleted instream detrital values. Isotopic niche partitioning was generally low among the minnow, chub, and shiner species across seasons whereas pupfish were more distinct. Notably, enriched δ15N values and constricted isotopic and diet niche areas from the spring to summer seasons suggested more specialized feeding habits for chub and shiner, likely due to increased resource availability and prey selectivity. Our study suggests that seasonal shifts in energy pathways and resource availability may disparately shape consumer feeding strategies. Evidence of spatiotemporal changes in food web structure may also provide a reference from which to compare how Red River consumers may be affected by habitat alteration and climate change.