Abstract: Fish diversity not only plays a pivotal role in maintaining the balance and stability of aquatic ecosystems, but also serves as a critical indicator for assessing the health status of freshwater ecosystems. However, prolonged anthropogenic disturbances, including hydraulic development, environmental pollution, and the introduction of exotic fish species, have resulted in varying degrees of habitat degradation for ichthyofauna in the Chongqing section of the Yangtze River Basin, posing severe threats to contemporary fish biodiversity. This study aimed to investigate the current status and spatial pattern evolution of fish diversity in the Chongqing section of the Yangtze River Basin following reservoir impoundment. From May to June 2022, environmental DNA (eDNA) technology was employed to survey fish diversity across six mainstream sections (from Jiangjin to Wushan) and eight important primary tributary sections. A total of 107 fish species were detected, belonging to 7 orders, 20 families, and 74 genera. These included 6 nationally protected species, 17 endemic species to the upper Yangtze River, and 12 exotic species. Comparative analysis of post-impoundment periods revealed an increase in exotic species compared to the trial operation phase of the Three Gorges Reservoir. Notably, the proportion of rheophilic fish species declined, while eurytopic fish species increased. Tributary habitats exhibited higher alpha diversity indices than adjacent mainstream sections. The grouping model demonstrated that the Jiangjin-Fuling section had a higher average alpha diversity index than the Fuling-Wushan section, with statistically significant differences in fish community composition between the groups. This study elucidates the current status and spatial pattern evolution of fish diversity in the Chongqing section of the Yangtze River Basin. It further validates the feasibility and efficiency of eDNA technology for fish diversity monitoring, positioning it as a robust complement to traditional survey methods.