Abstract: The Yuanjiang River is one of the most significant rivers in Yunnan, characterized by abundant fish resources; however, in recent years, human activities have led to a decline in these resources. This study employed environmental DNA (eDNA) technology to survey fish species in the Yuanjiang River, providing baseline data for fish resource management and conservation. In October 2023, 16 sampling stations were established along the river to collect water samples, and fish diversity was analyzed using eDNA techniques. A total of 47 fish species (5 orders, 15 families, and 40 genera) were detected, including rare and endangered species such as Bagarius rutilus and Hemibagrus guttatus, as well as 8 invasive fish species. Alpha diversity analysis revealed the following ranges across stations: Shannon-Wiener index (0.92–2.41), Simpson index (0.44–0.88), Pielou evenness index (0.35–0.60), and Margalef index (0.30–1.16). Hierarchical cluster analysis and non-metric multidimensional scaling (NMDS) were applied to examine spatial distribution patterns of fish communities, showing distinct differences among stations that grouped into three clusters from downstream to upstream, with geographically proximate stations clustering together. Similarity percentage analysis (SIMPER) identified species contributing to inter-group differences, with Culter alburnus, Channa gachua, Cirrhinus molitorella, and Abbottina rivularis as the primary contributors. Redundancy analysis (RDA) assessed relationships between fish diversity and environmental factors, indicating that the Yuanjiang River fish community is primarily influenced by altitude, salinity, conductivity, total dissolved solids, redox potential, and total phosphorus. This study demonstrates that eDNA technology is effective for analyzing fish species composition and distribution in the Yuanjiang River and can serve as a crucial tool for biodiversity monitoring and conservation.