In this article, a data matrix of 20 indicators (6960 observations) was obtained from 29 water quality monitoring stations of the Middle Route (MR) of the South-to-North Water Diversion Project of China (SNWDPC). Multivariate statistical techniques including analysis of variance (ANOVA), correlation analysis (CA), and principal component analysis (PCA) were applied to understand and identify the interrelationships between the different indicators and the most contributive sources of anthropogenic and natural impacts on water quality. The water quality index (WQI) was used to assess the classification and variation of water quality. The distributions of the indicators revealed that six heavy-metal indicators including arsenic (As), mercury (Hg), cadmium (Cd), chromium (Cr), selenium (Se), and lead (Pb) were within the Class I standard, while the As, Pb, and Cd displayed spatial variation. Moreover, some physicochemical indicators such as dissolved oxygen, 5-day biochemical oxygen demand (as BOD5), and total phosphorus (TP) had spatio-temporal variability. The correlation analysis result demonstrated that As, Hg, Cd, Cr, Se, Pb, copper (Cu), and zinc (Zn) had high correlation coefficients. The PCA result extracted three principal components (PC) accounting for 82.67% of the total variance, while the first PC was indicative of the mixed sources of anthropogenic and natural contributions, the second and the third PCs were mainly controlled by human activities and natural sources, respectively. The calculation results of the WQI showed an excellent water quality of the MR of the SNWDPC where the values of the stations ranged from 10.49 to 17.93, while Hg was the key indicator to determine the WQI > 20 of six stations, which indicated that the Hg can be the main potential threat to water quality and human health in this project. The result suggests that special attention should be paid to the monitoring of Hg, and the investigation and supervision within the areas of high-density human activities in this project should be taken to control the impacts of urban and industrial production and risk sources on water quality.