Abstract: The construction of the Three Gorges Dam altered hydrological regime, and impact on ecosystem and fish resources of the Yangtze River. The study applied the Ranges of Variability Approach (RVA) based on dividing hydrological periods to identify fish sensitive hydrological periods greatly influencing fish resources and assess the impact of key indicators of hydrologic alteration on fish resources in the middle typical reaches of the Yangtze River. The results showed that fish biomass of commercial fishes downstream of the Three Gorges Dam were greatly affected by the changes of discharge during flow rising period (March-June) at Yichang reach. The fry of four major Chinese carps were greatly affected by the changes of discharge during drying period (December-February) at Jianli reach. The fishery fish catch of the Dongting Lake were greatly affected by the changes of discharge during flooding period (July-September) near reaches of the Dongting Lake. The fishery fish catch of the Poyang Lake were greatly affected by the fluctuation of water level during retreating period (October-November) near reaches of the Poyang Lake. During these hydrological periods, the changes of key hydrological indicators, which describe extreme annual flow and duration, the change rate and frequency of flow had the greatest influence on fish resources in the middle typical reaches of the Yangtze River. Compared with traditional RVA without dividing hydrological periods, our study could identify the fish sensitive hydrological period and more indicators of hydrological alteration (IHAs) that greatly affect fish resources, also could quantify their impact on fish resources. The results could provide scientific thesis for fishery resources protection in the Yangtze River and the optimal operation of the Three Gorges Reservoir.