Simple SummaryThe water quality of reservoirs in water transfer projects is key as these reservoirs provide water directly to people. In general, the composition and structure of fish assemblage can reflect reservoir water quality and can also be regulated for its improving. We compared monitoring results of fish assemblages carried out by the traditional and environmental DNA methods in three-terminal reservoirs of the South-to-North Water Transfer project in China. The fish assemblages determined by both methods showed similar assemblage structure and patterns of diversity and spatial distribution; however, obvious differences in fish composition across the three reservoirs examined were found. Demersal and small fishes were the most common in all sites. Moreover, a strong association between water transfer distance and the distribution of non-native fish was found. Our findings highlight the importance of fish assemblage monitoring and the impact of water transfer distance on fish assemblage, providing valuable information that can be used in future water transfer projects.AbstractThe terminal reservoirs of water transfer projects directly supply water for domestic, agricultural, and industrial applications, and the water quality of these reservoirs produce crucial effects on the achievement of project targets. Typically, fish assemblages are monitored as indicators of reservoir water quality, and can also be regulated for its improvement. In the present study, we compared traditional fish landing (TFL) and environmental DNA (eDNA) metabarcoding methods for monitoring fish assemblages in three terminal reservoirs of the East Route of the South-to-North Water Transfer Project, China. Results of TFL and eDNA showed similar assemblage structures and patterns of diversity and spatial distribution with obvious differences in fish composition across three examined reservoirs. Demersal and small fish were dominant in all reservoirs. In addition, a strong association between water transfer distance and assemblages and distribution of non-native fish was found. Our findings highlight the necessity of the fish assemblage monitoring and managing for water quality and revealed the impact of water diversion distance on the structure of fish assemblages and dispersal of alien species along the water transfer project.