The mechanisms of biodiversity formation and maintenance (i.e., community assembly) have been an important topic in ecological research. The knowledge regarding biodiversity pattern and maintenance mechanisms of aquatic organisms in subtropical large rivers is still limited. In this study, the spatio-temporal diversity patterns of macroinvertebrate and their underlying mechanisms were systematically studied through a three-year survey (2015-2017) in the upper reaches of the Hanjiang River, the water source area of the middle route of the South-to-North Water Diversion Project. A total of 333 taxa were identified, belonging to 6 phyla, 10 classes, 25 orders and 110 families. Among them, Insecta was the species-richest group (278), followed by annelids (25), molluscs (19), malacostracous (7) and other animals (4). There were significant differences in taxa richness along spatial and temporal scales. Much more taxa were discovered in spring (227) than in summer (140), autumn (141) and winter (144). The near pristine Jinshui River harboured the most abundant taxa (179), while Si (112), Yue (149), Qi (140) and Jinqian (163) Rivers that were experiencing human disturbances, had much lower taxa richness. Distance based redundancy analysis (db-RDA) showed that both environmental variables and spatial factors were key drivers of community assembly, although the number and identity of these variables varied among seasons. Variation partitioning analysis suggested that environmental filtering played a more important role on community variation than spatial structuring. This study can provide important basis and reference for macroinvertebrate diversity assessments and environmental monitoring in this river basin.