Diatoms, microscopic unicellular algae, have been widely sampled from streams around the world because of their use in biological assessments, but as a result they are often only collected from epilithic habitats in riffles, which may lead to a substantial underreporting of their diversity and distributional patterns. The goals of this study were to identify how sampling methods and water chemistry affect the characterization of local and regional diversity and distributions. Species richness, Shannon diversity, and evenness were significantly greater in multiple habitat (MH) than epilithic habitat (EH) samples (paired t-test; P \ 0.01). At the local scale, MH sampling yielded 76% of the total species collected (42% unique), EH sampling captured 58% (24% unique), and 34% were shared. From MH samples, 3, 28, and 60 regionally rare species were collected from[75,[50, and [25% of sites, respectively, whereas 1, 14, and 34 were collected from EH samples, respectively. Regional abundance more strongly predicted site frequency than mean local abundance. Smaller drainage basins tended to have fewer species, likely because of species-area relationships, but local factors (% EH and alkalinity) influenced the taxonomic complexity of assemblages. Diversity was greatest at intermediate % EH, but low alkalinity reduced diversity, which is potentially important for ecosystems affected by anthropogenic acidification, as in our study. Multiple habitats need to be sampled for better documenting diatom diversity and distributions, which could improve conservation efforts at local and regional scales along with the characterization of ecological patterns and processes.