Agricultural non-point source pollution (ANPSP) is a primary cause of watershed water quality deterioration, and over 50% of NPS pollutants are estimated to come from ANPSP. Based on the “source-sink” theory and minimum cumulative resistance (MCR) model, ANPSP source and key resistance factors were integrated to identify areas at risk of ANPSP production and transportation into the waters of the upper Yangtze River basin. The results showed a spatial difference in the agricultural pollution sources of the basin, which were determined using both ANPSP loads and land-use types. Soil type, rainfall erosivity, and elevation were the three most important resistance factors in pollution transportation, weighting 0.373, 0.241, and 0.147, respectively. There was a spatial effect on the comprehensive resistance of ANPSP transportation, which was lower in mountainous terrain at the central basin. On the coupling of source and resistance processes, regions at serious risk of ANPSP were found to be concentrated in the southwest area. Areas at very high risk of NH₃-N and TP pollution accounted for 37.6% and 38.1%, respectively, in the total town/street area. The spatial risk patterns identified in this study could be used for decision making and policy regulation of ANPSP and for aquatic environmental protection.