Dam building and reservoir operations alter the downstream hydrological regime, and as a result, affect the health of the river aquatic ecosystem, particularly for large-scale cascade reservoirs. This study investigated the impact of the Gezhouba Reservoir (GR) and the Three Gorges Reservoir (TGR) on the spawning conditions of two critical taxa, i.e., the endemic four major carps and the endangered Chinese sturgeon in the Yangtze River. We analyzed the flow, sediment, and thermal regime in these two taxa spawning seasons and compared their features between the predam and postdam periods. Our results revealed that the GR and the TGR had altered the frequency distributions of flow, sediment, and water temperature to different degrees, with the impact by the GR on the carps and Chinese sturgeon ranked as water temperature > flow, sediment > water temperature > flow, and the effect of the TGR on these two taxa were ordered as flow > water temperature, sediment > flow > water temperature. For the GR, the satisfying degree of the suitable flow and water temperature of the carps increased, whilst the suitable flow, sediment, and water temperature for the Chinese sturgeon decreased. These changes in TGR showed a significant ascending (descending) trend in the suitable flow (water temperature) for the carps, and a clear decreasing trend in the flow, sediment, and temperature for Chinese sturgeon. Both the TGR and the GR had negative impacts on the spawning of these two taxa in terms of the rising/falling flow characteristics.