Abstract: Environmental DNA (eDNA) technology has significant advantages and potential in aquatic ecological monitoring of river and lake ecosystems, particularly in karst river basins. In order to understand the spatial distribution patterns of the benthic faunal communities and the environmental driving factors in the karst environment, this study focused on the Guizhou section of the Wujiang River Basin. We collected benthic fauna and water environment samples from 20 sites across the upstream and downstream reaches in August 2024. Benthic fauna were identified using eDNA metabarcoding technology. Furthermore, we employed species composition analysis, principal coordinate analysis (PCoA), Mantel tests, and co-occurrence network analysis to explore benthic faunal diversity, community structure, and their relationships with environmental factors. The results showed that a total of 107 benthic species were identified in the Wujiang River Basin, belonging to 4 phyla, 12 classes, 29 orders, 51 families, and 82 genera, with Arthropoda as the dominant phylum. There were significant differences in benthic community structure between the upstream and downstream reaches (R²=0.153, p=0.004), driven by key species including Philaccolilus mekus, Nais christinae, and Limnocythere sp. The permanganate index (COD_Mn) and flow velocity were the dominant environmental factors affecting the benthic community composition in the Wujiang River. Co-occurrence network analysis indicated that species interactions in upstream communities were maintained by multiple species, leading to more stable community function and structure. In contrast, downstream communities showed enhanced functional differentiation, with species interactions mainly relying on a few key taxa and lower stability. The study reveals significant spatial differentiation in the community structure and stability of benthic communities between the upstream and downstream reaches of the Wujiang River, and the results can provide a scientific reference for aquatic ecological protection and zonal management in the basin.