The diatoms are an ecologically important group of algae that have been extensively studied by ecologists and taxonomists. However, the large-scale patterns of diatom distribution and the factors underlying this distribution are largely unknown. The aims of this study were to identify the large-scale spatial patterns of benthic diatom assemblages in Korean streams and rivers, and to assess the importance of numerous environmental factors on diatom distribution. We classified 720 study sites based on diatom flora. Benthic diatoms, water chemistry, altitude, and riparian land cover and use were characterized by multivariate analyses, Monte Carlo permutation tests, and indicator species analysis. In total, we identified 531 diatom taxa. Diatom assemblages were mostly dominated by species of the genera Achnanthes, Navicula, Nitzschia, Cocconeis, Fragilaria (Synedra included), Cymbella, Gomphonema, and Melosira. Cluster analysis partitioned all 720 sites into eight groups based on diatom species composition. Canonical correspondence analysis indicated that altitude, land cover and use, current velocity, electrical conductivity, and nutrient levels explained a significant amount of the variation in the composition of assemblages of benthic diatoms. At the national scale, a downstream ecological gradient was apparent, from fast-flowing, mostly oligotrophic highland streams to slow-flowing, mostly eutrophic lowland rivers. Our data suggest that spatial factors explain some of the variation in diatom distribution. The present investigation of the spatial patterns of benthic diatoms, the ecological determinants of diatom occurrence, and the identification of diatom indicator species contributes to development of a program for assessing the biological integrity of lotic ecosystems in Korea.