In order to provide scientific supports for rivers ecological management of the Three Gorges Reservoir, we conducted a study to determine the spatial and temporal differentiation of water quality in the Gufu River. Gufu River, a tributary of Xiangxi River flowing into Three Gorges Reservoir, passes Shennongjia Forest District and Xingshan County, Hubei Province, Central China. We arranged 20 sampling sites along the Gufu River and measured 9 water quality parameters once a month from August 2010 to July 2011 at these sites. According to landscape difference, the sampling sites were divided into 4 groups: forest area (Group A), cultivated land area (Group B), reservoir area (Group C), village/small town area (Group D). According to hydrological rhythm, May to August were classed as wet season, and the other months were classed as dry season. The 9 water quality parameters were: total nitrogen (TN), dissolved oxygen (DO), total organic carbon (TOC), total phosphorus (TP), silicon dioxide (SiO₂), electrical conductivity (EC), chemical oxygen demand (COD), total hardness(T-Hard)and total alkalinity (T-Alk). Using the Discriminant Analysis (DA) and Analysis of Variance (ANOVA), we found the water quality showed spatial and temporal differentiation pattern: TP, SiO₂, EC, COD, T-Hard and T-Alk are significantly different among the different spatial group; TN, COD, DO, SiO₂ and TOC are different among the seasons; all water quality parameters presented significant seasonal changes at different spatial types. Using the Principal Component Analysis (PCA), we found the factor determining the water quality at different part of the river in different seasons. At the forest area (Group A), SiO₂ and TOC were the main factors which affect the water quality in the wet seasons, but in the dry seasons TP, SiO₂, TOC and COD were the mainly factors, which indicated that in forest area water quality was influenced by the natural factor such as surface runoff, vegetation and weathering. At the cultivated land area (Group B), in the wet season, T-Hard and TOC were the main factors affecting water quality while in the dry season TP, COD, T-Hard and T-Alk were the main factors affecting water quality. In cultivated land area, the variation of water quality was not only caused by the influence of natural factors, but also by factors of human activities such as agricultural runoff and domestic sewage without treatment. At reservoir area(Group C), SiO₂, TN and TP were the main factors affecting water quality in the wet season, while TN, TOC and SiO₂ were the main factors in the dry seasons. The water quality of the stream beside the villages/small towns was influenced by nitrogen and phosphorus particle entering into reservoir with farmland runoff, surface runoff and untreated domestic sewage. At village/small town area (Group D), SiO₂, TN and TP were the main factors affecting water quality in the wet season, while T-Alk, T-Hard, TN and TOC were the main factors in the dry seasons. The water quality at the village/small town area was influenced by municipal sewage, agricultural runoff. Our study indicated that the water quality of Gufu River is affected by natural and anthropogenic factors and the later one are greater. Therefore, the strengthening of the management and protection of water resources and good long-term water quality monitoring is particularly important for the improvement of water quality.