The paradigm of tight pelagic-benthic coupling in the Arctic suggests that current and future fluctuations in sea ice, primary production, and riverine input resulting from global climate change will have major impacts on benthic ecosystems. To understand how these changes will affect benthic ecosystem function, we must characterize diversity, spatial distribution, and community composition for all faunal components. Bacteria and archaea link the biotic and abiotic realms, playing important roles in organic matter (OM) decomposition, biogeochemical cycling, and contaminant degradation, yet sediment microbial communities have rarely been examined in the North American Arctic. Shifts in microbial community structure and composition occur with shifts in OM inputs and contaminant exposure, with implications for shifts in ecological function. Furthermore, the characterization of benthic microbial communities provides a foundation from which to build focused experimental research. We assessed diversity and community structure of benthic prokaryotes in the upper 1 cm of sediments in the southern Beaufort Sea (United States and Canada), and investigated environmental correlates of prokaryotic community structure over a broad spatial scale (spanning 1,229 km) at depths ranging from 17 to 1,200 m. Based on hierarchical clustering, we identified four prokaryotic assemblages from the 85 samples analyzed. Two were largely delineated by the markedly different environmental conditions in shallow shelf vs. upper continental slope sediments. A third assemblage was mainly comprised of operational taxonomic units (OTUs) shared between the shallow shelf and upper slope assemblages. The fourth assemblage corresponded to sediments receiving heavier OM loading, likely resulting in a shallower anoxic layer. These sites may also harbor microbial mats and/or methane seeps. Substructure within these assemblages generally reflected turnover along a longitudinal gradient, which may be related to the quantity and composition of OM deposited to the seafloor; bathymetry and the Mackenzie River were the two major factors influencing prokaryote distribution on this scale. In a broader geographical context, differences in prokaryotic community structure between the Beaufort Sea and Norwegian Arctic suggest that benthic microbes may reflect regional differences in the hydrography, biogeochemistry, and bathymetry of Arctic shelf systems.