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We examined inter-habitat variation in benthic macroinvertebrate assemblages in the 180-km Garrison Reach of the Upper Missouri River, North Dakota (USA) in 2001–2003. The Garrison Reach is unchannelized with a mostly rural setting. Flows are regulated by Garrison Dam. We sampled benthos from three habitats defined a priori: channel, shoreline, and backwater. Benthic assemblages were different in each habitat. Average Bray-Curtis dissimilarity in assemblage composition ranged from 89% for backwater versus channel habitat to 70% for backwater versus shoreline habitat. There were distinct intra-habitat groups within a priori habitats: channel assemblages included moving-sand assemblages and other-substrate channel assemblages; backwater assemblages included connected (to the river channel) and unconnected backwater assemblages; shorelines assemblages varied between natural (unprotected) and riprap (rock revetment) shorelines. Abundance and taxa richness were lowest and spatial variability highest for moving-sand channel assemblages. Abundance was highest in backwaters. Taxa richness in backwaters and along channel shorelines were similar. Assemblages in all three habitats were dominated by Nematoda, Oligochaeta and Chironomidae. Taxa in these groups comprised at least 80% of mean abundance in all three habitats. Taxa that discriminated among habitats included the psammophilic chironomid Chernovskiia for moving-sand channel substrates versus all other habitats; Hydroptila (Trichoptera) for riprap vs natural shorelines, Aulodrilus (Oligochaeta) for connected versus unconnected backwaters; and Nematoda for backwater versus channel and shoreline versus channel. Based on overlap patterns in benthic assemblages among habitats, we concluded that sampling main channel shorelines should also capture much of the natural and stressor-induced variation in connected backwater and channel habitat exclusive of moving-sand channel habitat.