Abstract: We used environmental DNA metabarcoding to survey fish communities in the protected area across three seasons—summer (August 2024), winter (December 2024), and spring (April 2025)—and examined their spatiotemporal variation in relation to multiple diversity dimensions and environmental drivers; in total, we recorded 139species belonging to 7 orders, 21 families, and 89 genera, including 10 nationally protected species, 46 endemics to the upper Yangtze River, and 11 non-native species, with species richness peaking in winter (116species). Clear spatiotemporal heterogeneity emerged among the fish communities: the Minjiang River (MJR) consistently showed the highest diversity and niche breadth across all seasons and especially in summer, the Chishui River (CSH) stood out in winter, and the Yangtze River (YZR) displayed a distinctive pattern of richness differentiation. Species turnover accounted for over 90% of beta diversity, making it the dominant process shaping community differences, with the strongest turnover occurring in winter. As for environmental associations, elevation and total nitrogen correlated positively with niche breadth and functional richness, and may have indirectly influenced species turnover; in contrast, water temperature, total organic carbon, and total phosphorus were negatively correlated with Rao’s quadratic entropy and functional richness, potentially favouring increased nestedness, while latitude generally showed negative correlations with both species diversity and niche breadth. Overall, stochastic processes appeared to govern community assembly, although deterministic processes also played notable roles in specific regions and seasons. Our findings offer empirical evidence for the mechanisms that sustain multidimensional fish diversity in the reserve, and provide a useful reference for the conservation and management of fish resources in the upper Yangtze River.