To identify indicator species of aquatic macrophytes and elucidate their responses to environmental gradients and spatial processes in typical tributaries of the middle and lower Yangtze River, we investigated the Xiangjiang, Hanjiang, and Ganjiang Rivers using environmental DNA (eDNA) metabarcoding combined with water-quality, bioclimatic, and topographic variables. Community structure, indicator species distribution patterns, and their driving mechanisms were systematically analyzed.The results revealed significant differences in aquatic macrophyte community composition among the three tributaries. After controlling for spatial structure, ammonium nitrogen remained the only significant environmental variable associated with community variation. Variation partitioning showed that the independent contribution of spatial variables exceeded that of environmental variables, and significant residual spatial autocorrelation was detected, indicating an important role of spatial processes in community assembly.Indicator species analysis (IndVal) identified three significant taxa, including Stuckenia pectinata in the Xiangjiang River and Typha latifolia and Trapa bicornis in the Hanjiang River. Generalized linear models (GLM) and random forest (RF) models consistently demonstrated species-specific responses to environmental and spatial factors. S. pectinata was primarily associated with the ammonium-nitrogen gradient, whereas T. latifolia was mainly structured by spatial variables. T. bicornis responded to both environmental and spatial factors but exhibited relatively weak explanatory relationships. Both modeling approaches showed satisfactory predictive performance, indicating that the integrated framework of environmental and spatial variables effectively explained indicator-species distribution patterns.In addition, although eDNA metabarcoding showed relatively low consistency with traditional field surveys, it exhibited strong complementarity and substantially expanded species detection coverage.Overall, our findings suggest that the distribution of aquatic macrophyte communities and their indicator species is jointly shaped by environmental filtering and spatial processes rather than solely by local environmental conditions. Spatial structure plays a critical role in modulating environmental signals and influencing species distributions. This study provides a theoretical basis for indicator-species identification, understanding community assembly mechanisms, and applying eDNA metabarcoding in riverine ecological monitoring.