Conserving and enhancing freshwater biodiversity are global issues to ensure ecosystem integrity and sustainability. To meet this, it is critical to understand how the biological assemblages are determined by environmental gradients in different spatial scales. Nevertheless, information on their large-scale environmental relationships remains scarce in Korea. We aimed to understand nationwide spatial distribution patterns of benthic macroinvertebrates and important environmental factors affecting their distribution in 388 streams and rivers across Korea. A total of 340 taxa, belonging to 113 families in 23 orders of five phyla, were identified. Assemblage composition in most Korean streams included a few predominant colonizers and a majority of rare taxa. Cluster analysis based on benthic macroinvertebrates classified a total of 720 sampling sites into five clusters according to the pollution levels from fast-flowing less polluted streams with low electrical conductivity to moderately or severely polluted streams with high electrical conductivity and slow water velocity. Canonical correspondence analysis revealed that altitude, water velocity and streambed composition were the most important determinants, rather than watershed and water chemistry variables, for explaining the variation in macroinvertebrate assemblage patterns. The results provide basic information for establishing the conservation and restoration strategies of macroinvertebrate biodiversity against anthropogenic disturbances and developing more confident bio-assessment tools for diagnosing stream ecosystem integrity.