Abstract: Understanding the effects of hydrological connectivity loss on lake water quality is critical for developing targeted restoration strategies. As China’s largest freshwater and a historically river-connected lake, Poyang Lake has experienced progressive fragmentation since the 20th century due to dam and levee construction, isolating its subsidiary lakes (e.g., Zhuhu, Junshan, Kangshan, Xinmiao, and Nanbeigang). However, systematic comparisons of water quality between the main lake and these isolated subsystems remain scarce. To address this gap, we conducted a comprehensive survey analyzing spatiotemporal variations in physicochemical parameters. Key findings revealed: (1) Isolated lakes exhibited greater fluctuations than Poyang Lake, particularly for Chl-a (CV: 79% in Kangshan Lake vs. 73% in Junshan Lake) and nutrients (CODMn, NH3-N, PO4-P, TN, TP). (2) Kangshan Lake (γ = 1.25) and Nanbeigang (γ = 1.14) reached moderate pollution levels, significantly exceeding Poyang Lake in TN (p < 0.05 vs. Junshan/Zhuhu/Xinmiao) and TP (p < 0.05 vs. Zhuhu/Nanbeigang). (3) Anthropogenic pressures (fertilizer use, industrial density) dominated water quality disparities, while hydrological factors (water depth) exerted secondary effects. These results underscored the need for dual interventions: (i) stringent control of watershed pollutant loads and (ii) engineered reconnection to restore natural flow regimes.