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Abstract Fish are vital in river ecosystems; however, traditional investigations of fish usually cause ecological damage. Extracting DNA from aquatic environments and identifying DNA sequences offer an alternative, noninvasive approach for detecting fish species. In this study, the effects of environmental DNA (eDNA), coupled with PCR and next‐generation sequencing, and electrofishing for identifying fish community composition and diversity were compared. In three subtropical rivers of southern China, fish specimens and eDNA in water were collected along the longitudinal (upstream–downstream) gradient of the rivers. Both fish population parameters, including species abundance and biomass, and eDNA OTU richness grouped 38 sampling sites into eight spatial zones with significant differences in local fish community composition. Compared with order‐/family‐level grouping, genus‐/species‐level grouping could more accurately reveal the differences between upstream zones I−III, midstream zones IV−V, and downstream zones VI–VIII. From the headwaters to the estuary, two environmental gradients significantly influenced the longitudinal distribution of the fish species, including the first gradient composed of habitat and physical water parameters and the second gradient composed of chemical water parameters. The high regression coefficient of alpha diversity between eDNA and electrofishing methods as well as the accurate identification of dominant, alien, and biomarker species in each spatial zone indicated that eDNA could characterize fish community attributes at a level similar to that of traditional approaches. Overall, our results demonstrated that eDNA metabarcoding can be used as an effective tool for revealing fish composition and diversity, which is important for using the eDNA technique in aquatic field monitoring.