Abstract: It’s acknowledged that reclaimed water contains abundant inorganic nitrogen and phosphorus and has become an important supplementary water source for urban rivers and lakes. Investigating its effects on the spatial distribution of benthic chironomid communities is of great theoretical and practical significance for the scientific assessment of urban river ecosystem health. Based on these, the river reach near the reclaimed water outfall of the Tonghui River in Beijing was selected as the study area. By integrating DNA barcoding and community statistical analyses, including cluster analysis, non-metric multidimensional scaling, and discriminant analysis, the spatial distribution characteristics of riparian chironomid communities primarily replenished by reclaimed water were systematically analyzed and the spatial variation patterns of community structure at different taxonomic levels and their environmental driving mechanisms were revealed. The results showed that chironomid communities exhibited significant spatial differences across different taxonomic levels, with upstream sites being dominated by non-receiving water groups and downstream sites being dominated by receiving water groups. Moreover, the spatial differences between the two groups increased significantly from higher to lower taxonomic levels. At the subfamily level, Chironominae and Orthocladiinae were identified as key taxa characterizing the differences between the two groups. At the genus level, Dicrotendipes, Cricotopus, and Tanytarsus significantly discriminated between the two groups, while at the species level, Dicrotendipes pelochloris was identified as an indicator species distinguishing between them. Sediment pH, organic matter (OM), soil organic carbon (SOC), total nitrogen (TN) and total phosphorus, as well as water chemical oxygen demand, ammonia nitrogen (NH₃-N), and chlorophyll-a (Chl-a) concentrations, significantly influenced the spatial variation in chironomid community structure. In addition, sediment OM, SOC, and TN, together with water pH, TN, total organic carbon, NH₃-N, and Chl-a, had significant effects on the spatial variation in community diversity. Furthermore, Paratanytarsus, Cardiocladius, and Chironomus circumdatus were identified as indicator taxa reflecting the spatial variation in chironomid community structure along an environmental gradient from highly to less polluted conditions. These findings contribute to an improved understanding of the spatial variation patterns of chironomid community structure at different taxonomic levels under reclaimed water replenishment, as well as the driving mechanisms mediated by physicochemical properties of overlying water and sediments. They also provide a scientific basis for ecological health assessment and water environment management of urban river ecosystems.