Natural variability in hydrologic regimes and lack of empirical relationships between flow and ecological response have created challenges for instream flow protection and restoration. We performed a nationwide analysis of hydrologic alteration downstream of dams and associated changes in functional traits of aquatic insect assemblages to develop generalizable relationships between degree of flow alteration and ecological response. Sites (undammed and downstream of dams) with long-term records of mean daily flow at stream gages and aquatic insect data from the U.S. Geological Survey’s National Water Quality Assessment (NAWQA) Program were grouped into hydrologic classes (groundwater, perennial, snowmelt, and intermittent) and degree of flow alteration was examined. Undammed sites were assumed to have 0% deviation in flows. Dammed sites were paired with nearby reference (undammed) sites and percent deviation of flow metrics was calculated by comparing daily flow data for each pair of sites in the same hydrologic class, adjusted for basin area. We examined links between degree of hydrologic alteration (ranging from 0% to over 100%) and relative proportion of taxa and aquatic insect abundance in each functional trait category (e.g., occurrence in drift, size at maturity, voltinism) to establish stressor-response relationships between hydrologic variables and functional composition of aquatic insects.