The Kherlen River Basin is a typical basin in the eastern Mongolian Plateau and is dominated by grassland. This study estimated the grassland yield in the Kherlen River Basin using the Carnegie–Ames–Stanford approach (CASA) model, combined with Theil–Sen median trend analysis and the Geodetector, to explore its spatiotemporal changes and driving factors. This integrated framework links temporal trend detection with spatial interaction analysis to better reveal ecological responses to climatic and anthropogenic influences. The results showed the following: (1) The root mean square error (RMSE) between the estimated grassland yield and the laboratory measurements was 37.88 g/m², with an estimation accuracy (EA) of 73.52%. (2) From 2000 to 2024, the grassland yield increased significantly at a rate of 1.98 g/(m²·a) (p < 0.05), with the fastest growth in the middle reaches. (3) Spatially, 79.78% of the basin exhibited significant increases, mainly in the central and western regions. The proportion of significant increase was highest in the upper reaches (40.36%), followed by the middle (32.89%) and lower reaches (6.53%). (4) Due to limited temporal resolution of socioeconomic data, the driving factor analysis covered the period 2000–2020, during which the overall grassland yield was primarily influenced by the interaction between precipitation and elevation (q = 0.6371). Specifically, the upper, middle, and lower reaches were mainly influenced by the interactions between temperature and precipitation (q = 0.6772), precipitation and elevation (q = 0.6377), and temperature and elevation (q = 0.4255), respectively. The study indicates that grassland yield in the Kherlen River Basin exhibited an overall increasing trend during 2000–2024, with climatic factors (precipitation and temperature) and the geographic factor (elevation) identified as the dominant drivers. The influence of human activities was not significant, although this result may be affected by uncertainties associated with data resolution limitations. Future work should incorporate higher-resolution remote sensing and socioeconomic datasets to better assess the impacts of human activities.