Diatom assemblage structure on natural and artificial substrata was related to lotic physical habitat characteristics in an agricultural basin in Minnesota. We found 59 species across the basin, but species richness at individual sites ranged from 4 to 25 (median 16) on natural substrata and 9 to 23 (median 15) on artificial substrata. Most species were rare, with nearly half appearing at fewer than 3 sites. The most abundant taxa were biraphid diatoms (relative abundance >28% in most cases). Rank correlations of guild relative abundance from the artificial substrata (Achnanthes, biraphid, silt-tolerant) were strongest with % bank erosion and the Qualitative Habitat Evaluation Index riparian subscore, both of which were negatively correlated. Diatom assemblages from natural substrata showed strong rank correlations between % canopy coverage and the Achnanthes, biraphid, rheophilic, and silt tolerant guilds. Relative abundance of stalked diatoms and the width of the streams at flood were also strongly correlated. TWINSPAN, and subsequent Kruskal-Wallis testing of differences in habitat condition between the site groupings, provided similar results. Relative abundance of Molloy's Achnanthes, biraphid, and stalked guilds, and the relative abundance of silt tolerant taxa (Bahls's siltation index) appear to have potential for bioassessment of physical habitat in streams of agricultural drainages.