Abstract: Dongjiang Lake is an important drinking water source in Hunan Province and a strategic water source for Changsha-Zhuzhou-Xiangtan City Group. Zheshui River, as its upper level tributary, is an important ecological security barrier in Dongjiang Lake. Currently, under the background of active development of regional agricultural clusters such as vegetables, tea, fruits and ecological breeding, water environment status and pollution sources of Zheshui River are still unclear. Based on measured water quality data of basin, water quality assessment was conducted using water pollution index (WPI), water quality health assessment (ETI) and water quality index (WQI), and principal component analysis (PCA) to identify the main pollutants and key source areas of the basin. Finally, absolute principal component-multiple linear regression (APCS-MLR) model and output coefficient method were used to analyze the main pollution sources and their contribution rates. The results show that: (1) From 2016 to 2022, overall water quality of Zheshui River Basin showed a trend of fluctuating deterioration followed by gradual improvement, but concentration of total nitrogen (TN) exceeded the standard for a long time, belonging to Class Ⅳ water. TN was a key factor affecting water environment of Dongjiang Lake in the downstream. (2) The water quality of Zheshui River Basin was better in the upper and lower reaches, and worse in the middle reaches, especially the worst spatial pattern near the county seat, which indicate that water pollution of the basin was mainly affected by exogenous pollution. (3) The extracted three principal components could explain 88.77% of original variable information, organic pollutants (F1) accounted for 38.29%, nitrogen pollutants (F2) accounted for 34.86%, and phosphorus pollutants (F3) accounted for 15.62%. F1 mainly came from living source + agricultural non-point source (mainly livestock and poultry breeding); F2 mainly came from living source + agricultural non-point source (mainly planting), Luyang Town and Tuqiao Town were the key source areas; F3 mainly came from agricultural non-point source (mainly livestock and poultry breeding) + living source, and Maqiao Town and Quanshui Town were the key source areas. In the future, the precise control of agricultural non-point source pollution should be strengthened in key source areas of Zheshui River Basin, which was of great significance to protect the water environment of Zheshui River and Dongjiang Lake in the downstream.