The middle and lower Yangtze River floodplain lakes are important stopover and wintering sites for migratory birds on the East Asian–Australasian Flyway. The periodic water level fluctuation of the lakes plays a critical role in the aggregation of waterbirds. However, the construction of a series of gates and dams has impeded the hydrological connectivity between rivers and lakes and disrupted the natural water level fluctuation patterns of lakes. The water level status of lakes influences habitat utilization by waterbirds. In recent years, Caizi Lakes, the gate-controlled lakes connected to the Yangtze River due to the need for water level regulation requirements, has experienced sustained high water levels in winter, which can reduce habitat quality and in turn influence waterbirds activity. In the present study, wintering waterbirds in Caizi Lakes were surveyed during the 2021–2022 (W1) and 2022–2023 (W2) wintering periods. Moreover, we explored the effects of constant high water levels in winter on waterbird assemblages by comparing species richness, abundance, and functional alpha and beta diversity. Correlation analyses were then performed to elucidate the relationship between habitat factors and water level changes during the wintering period of migratory waterbirds and between changes in various habitat factors and wintering waterbird assemblages under different hydrological conditions. The results showed that functional beta diversity was more sensitive to changes in waterbird assemblages in Caizi Lakes than functional alpha diversity. Moreover, constant high water levels in the mid-wintering period decreased suitable habitats for wintering waterbirds, increased functional turnover among waterbird assemblages, increased total dissimilarity among assemblages significantly, and decreased the level of functional nestedness significantly. Additionally, the areas of the three wetland habitats in Caizi Lakes were significantly correlated with water level changes during W1 and relatively less affected by water level changes under constant high water level conditions during W2. The changes in habitat area were influenced primarily by anthropogenic activities, with an increase in meadow area favorable for species richness and abundance, as well as functionally nested components. Water level regulation should take into consideration the natural hydrological rhythms of the declining water level to ensure that adequate and suitable habitats are provided for wintering waterbirds.