Abstract: The Heishui River is an important alternative habitat for fish in the mainstream of the lower Jinsha River within the Baihetan Reservoir area and is a priority-protected tributary. To explore a method for surveying fish diversity in the Heishui River, fish diversity was assessed using environmental DNA metabarcoding (eDNA) technology on samples collected from 11 sections along the lower reaches of the Heishui River in November 2024 and May 2025. The results showed that: ① Through environmental DNA sample collection and high-throughput sequencing analysis, a total of 57 fish species were detected, belonging to 4 orders, 14 families, and 40 genera. Among them, Cypriniformes were the most abundant, with 39 species. The relative OTU abundance of Cypriniformes was the highest (76.45%), followed by Perciformes (18.87%) and Siluriformes (4.68%). The dominant species were Schistura fasciolata, Cyprinus carpio, Rhinogobius similis, Zacco platypus, Schizothorax kozlovi, Oreochromis niloticus, and Chanodichthys erythropterus.② A total of eight endemic fish species of the upper Changjiang River were detected, including Schizothorax wangchiachii and Schizothorax kozlovi, which were present in all survey sections. Five alien species were found, and Oreochromis niloticus was abundantly detected in all survey sections during both spring and autumn.③ Diversity analysis showed no significant difference in diversity indices between autumn and spring. Section H11 had the highest number of species, with a fish composition distinct from other sections. These results provide detailed data on fish diversity in the lower Heishui River, confirm the feasibility of using eDNA technology to investigate fishery resources in the basin, and suggest that when integrated with traditional survey methods, environmental DNA technology can serve as a crucial supplementary tool for rapidly understanding the spatiotemporal dynamics of fish resources within a watershed. The research can provide important technical support for the monitoring and protection of fish resources in the basin.