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The complex water regimes and fragile ecological systems in Dongting Lake and Poyang Lake, located in the middle reach of the Yangtze River, have been significantly affected by regional climate change and anthropogenic activities. The hydrological data from the outlets of Dongting Lake (Chenglingji station) during 1955-2016 and Poyang Lake (Hukou station) during 1953-2014 were divided into two periods: the pre-impact period and the post-impact period. Four statistical tests were used to identify the change years: 1979 at Chenglingji and 2003 at Hukou. The indicators of hydrologic alteration and range of variability approach were used to assess alterations in water level regimes. Results show that the severely altered indicators were January water level at both lake outlets, and 1-, 3-, 7- and 30-day minimum water level at Chenglingji, with the degree of hydrological alteration being larger than 85%. The overall degrees of hydrological alteration at Chenglingji and Hukou were 52.6% and 38.2%, respectively, indicating that water level regimes experienced moderate alteration and low alteration or that ecosystems were at moderate risk and low risk, respectively. Changes in water level regimes were jointly affected by climate change and anthropogenic activities. Water level regimes at Dongting Lake outlet were mainly affected by increased rainfall and dam regulation. Decreased rainfall, dam regulation, and sediment erosion and deposition were the main impact factors of water level regimes at Poyang Lake outlet. These changes in water level regimes have greatly influenced both aquatic and terrestrial ecosystems, especially for fish and vegetation communities. This study is beneficial for water resource management and ecosystems protection under regional changes.