Macrophytes are affected by many natural and human stressors globally but their long-term responses to these multiple stressors are not often quantified. We employed remote sensing and statistical tools to analyze datasets from both short-term (2017–2018) field investigations to explore seasonal patterns, and long-term (1988–2018) Landsat remote-sensing images to detect annual patterns of macrophyte distributions and study their responses to changes in climate, hydrology, and anthropogenic activities in a chain of water diversion lakes in eastern China. We found: 1) biomass and species richness of macrophytes peaked in summer with dominant species of submerged macrophytes Ceratophyllum demersum, Potamogeton pectinatus, and Potamogeton maackianus and floating macrophytes Trapa bispinosa, and non-native species Cabomba caroliniana spread in midstream Luoma Lake and Nansi Lake in summer, while Potamogeton crispus was dominant in all the lakes in spring; 2) water physicochemical parameters (chloride and water depth), lake characteristics (area and water storage), climate factors (air temperature and precipitation), and anthropogenic activities (commercial fishery and urban development) were significantly correlated to the seasonal distribution of macrophytes; 3) long-term data showed a significantly negative correlation between coverage of floating macrophytes and precipitation where the wettest year of 2003 had the lowest coverage of floating macrophytes; and 4) climate (air temperature) and hydrology (water level) were positively correlated with total macrophyte coverage, but human disturbance indexed by the gross domestic product was negatively driving long-term coverage of macrophytes. Our study has important implications for understanding the long-term succession of macrophytes under both natural and human stressors, and for future environmental management and ecological restoration of freshwater lakes.