Rivers possess unique and complex branching hierarchical network topology, which leads to noticeable differences in connectivity and habitat heterogeneity across different sections of the river network. Historically, river ecosystems have been studied as local or linear structures, overlooking the influence of river network positions on ecological phenomena, such as community assembly. We focused on the Chishui River, which has a complex network structure and natural flow regime, to explore the effects of river network positions on species diversity (α and β diversity) and community assembly of macroinvertebrates. α diversity was measured using species richness, Shannon-Wiener diversity index, Simpson diversity index, and Pielou evenness index. β diversity was characterized using the Sørensen dissimilarity index (β_sør) and Bray-Curtis dissimilarity index (d_BC), which were further partitioned into turnover and nestedness components, respectively. Community assembly was assessed using Redundancy analysis (RDA) and associated variation partitioning. We found that: 1) Macroinvertebrate communities in the entire river basin are primarily regulated by environmental filtering. 2) River network positions affected the diversity patterns and community assembly processes in the Chishui River. Mainstream communities were primarily influenced by environmental filtering, while tributary communities were structured by both spatial constraints and environmental filtering. These findings challenge the applicability of the Network Position Hypothesis (NPH) in the Chishui River Basin. Additionally, α diversity was significantly higher in tributaries compared to the mainstream, whereas β diversity was significantly higher in the mainstream. β diversity of macroinvertebrate communities was mainly composed of turnover, suggesting that protecting different sites and habitats is crucial for maintaining regional species diversity. This study provides insights into the mechanisms of species coexistence and maintenance in benthic communities under the influence of complex river networks and contributes to the development of new hypotheses applicable to river networks in subtropical monsoon regions in China. It also provides reference and basis for site selection and design of biological protection zones in the upper reaches of the Yangtze River.