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Determining the patterns of diversity and community composition in headwater streams is fundamental to river biodiversity conservation. Environmental selection has been assumed to be a major force driving temperate macroinvertebrate assembly. To test this assumption in the subtropics, we conducted identical surveys for headwater streams in two neighboring basins, which are located on two sides of a north–south mountain in southern China. We sampled macroinvertebrates and measured habitat and environmental variables in headwater streams of the two basins. The two groups of headwater streams share a species pool and have similar taxa, taxa richness, and functional composition. Beta diversity accounted for a high proportion of the within-basin diversity, and was mainly attributed to the replacement differences. Highly similarity between the two basins' species composition showed similar environmental selection in structuring macroinvertebrate communities at the regional scale. Redundancy analysis showed that basin identity is the key factor explaining the variation of communities. Environmental selection is an important factor in structuring macroinvertebrate communities within individual basins. Annual precipitation differs significantly on the two sides of the mountain shaded by the East Asia monsoon, resulting in distinctive substrate compositions in the two basins. Our study provides empirical support for the roles of environmental selection in shaping subtropical macroinvertebrate communities. [ABSTRACT FROM AUTHOR]