Lake Yutan is one of the four main water supply initiatives in western Chongqing, with two inflow rivers. It is a typical multi-habitat ecosystem. To date, the fish composition and diversity in Lake Yutan have been scarcely reported. In this study, environmental DNA (eDNA) technology combined with the traditional fish survey was used to investigate the fish community and diversity in Lake Yutan, aiming to explore the potentials of eDNA technology in assessing fish diversity within river-reservoir habitats. The results showed that a total of 36 fish species categorized into 4 orders, 11 families, and 32 genera were identified by eDNA technology. The analysis of relative sequence abundance revealed higher relative abundance for Hypophthalmichthys nobilisand Hypophthalmichthys molitrix . A total of 21 fish species, categorized into 3 orders, 9 families, and 20 genera, were captured by the traditional fish survey. A total of 42 fish species were detected using the eDNA technology and traditional fish survey, with 15 species being jointly investigated, accounting for 71.43% of the total fishes captured through the traditional fish survey. The results of αand βdiversity analysis based on sequence abundance revealed variations in fish composition and diversity across the lake region, estuary, and the buffer zone at the inlet. The fish diversity in the estuary and inlet buffer zone were higher than that observed in the lake region. In summary, our findings demonstrated that the application of eDNA technology had a superior detection rate for fish species compared with the traditional fish survey, and thereby could serve as an invaluable non-invasive supplementary tool for monitoring fishery resources and enhancing the reliability of river-reservoir habitat resource survey.