Prokaryote communities were investigated on the seasonally stratified Alaska Beaufort Shelf (ABS). Water and sediment directly underlying water with origin in the Arctic, Pacific or Atlantic oceans were analyzed by pyrosequencing and length heterogeneity-PCR in conjunction with physicochemical and geographic distance data to determine what features structure ABS microbiomes. Distinct bacterial communities were evident in all water masses. Alphaproteobacteria explained similarity in Arctic surface water and Pacific derived water. Deltaproteobacteria were abundant in Atlantic origin water and drove similarity among samples. Most archaeal sequences in water were related to unclassified marine Euryarchaeota. Sediment communities influenced by Pacific and Atlantic water were distinct from each other and pelagic communities. Firmicutes and Chloroflexi were abundant in sediment, although their distribution varied in Atlantic and Pacific influenced sites. Thermoprotei dominated archaea in Pacific influenced sediments and Methanomicrobia dominated in methane-containing Atlantic influenced sediments. Length heterogeneity-PCR data from this study were analyzed with data from methane-containing sediments in other regions. Pacific influenced ABS sediments clustered with Pacific sites from New Zealand and Chilean coastal margins. Atlantic influenced ABS sediments formed another distinct cluster. Density and salinity were significant structuring features on pelagic communities. Porosity co-varied with benthic community structure across sites and methane did not. This study indicates that the origin of water overlying sediments shapes benthic communities locally and globally and that hydrography exerts greater influence on microbial community structure than the availability of methane.