The Danjiangkou Reservoir is a key water source for the South-to-North Water Diversion Project"s middle route and a national first-level water source protection zone. Since water began flowing in 2014, the overall water quality in the reservoir area has remained at Class II or higher, playing an important ecological and water resource protection role. Water level fluctuations, as a crucial parameter in reservoir operation, directly affect the environmental characteristics of the water body, thereby indirectly influencing the growth, distribution, and diversity of phytoplankton. To investigate the impact of habitat changes induced by water level fluctuations on the functional groups of phytoplankton in the Danjiangkou Reservoir, this study selected high-water periods (October–December 2023) and low-water periods (April–June 2024) for sampling and analysis. The study analyzed the structure and composition of phytoplankton functional groups and the environmental factors influencing these groups. The results showed that a total of 128 species belonging to 75 genera and 7 phyla of phytoplankton were identified under different temporal and spatial conditions in the Danjiangkou Reservoir. The main phytoplankton phyla were Bacillariophyta (39.06%), Chlorophyta (35.94%), and Cyanophyta (10.16%). There were significant differences in the composition of phytoplankton functional groups between different temporal and spatial conditions (p < 0.05). A total of 26 functional groups were classified, including functional groups N and P that adapt to mixed water column environments, functional groups Y and T adapted to still water environments, and universal functional groups M and Lo. During the high-water period, the dominant functional group in the reservoir area was group B; while during the low-water period, the dominant functional groups were MP, P, and Lo. In the tributaries, the dominant functional groups during the high-water period were M, Lo, and S1, while during the low-water period, the dominant functional groups were TC and B. The results of the Redundancy Analysis (RDA) indicated that pH, turbidity, NO3-N, and PO43--P were the main environmental factors affecting the dominant functional groups of phytoplankton. Water quality evaluation based on the Q-index showed that the water quality in the reservoir area was generally in a "good" state, while the water quality in the tributary area ranged from "poor" to "good." The study suggests that while the water quality in the main reservoir area is generally stable, the tributaries experience significant interference from nutrient pollution on phytoplankton communities during the high-water period, highlighting the need for reservoir management to focus on the potential impact of external inputs during high-water periods on the ecosystem.