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The health of freshwater biota is dependent on streamflow, yet identification of the flow regimes required to maintain ecological integrity remains challenging to states in the United States seeking to establish ecological flows. We tested the relationship between decreases in streamflow and Shannon-Weaver diversity index of fish species for four flow-based habitat guilds: riffle, riffle-run, pool-run, and pool in North Carolina. We found species that prefer shallow habitats, such as riffles and riffle-runs were the most sensitive to decreases in streamflow; whereas no significant relationships were found for pool or pool-run species. The sensitivity to decreases in streamflow was greatest during summer and fall, when streams are naturally lower. When all fish habitat guilds were included in the assessment of flow-biology relationships, there were no significant relationships to decreases in streamflow. As the sensitivity of fish to reductions in streamflow is not constant across habitat guilds, combining all fish species together for flow-biology analyses may greatly underestimate the response of fish species to decreases in flow and should be acknowledged when establishing ecological flows.