Hydrothermal vents differ both in surface input and subsurface geochemistry. The effects of these differences on their microbial communities are not clear. Here, we investigated both alpha and beta diversity of diffuse flow-associated microbial communities emanating from vents at a basalt-based hydrothermal system along the East Pacific Rise (EPR) and a sediment-based hydrothermal system, Guaymas Basin. Both Bacteria and Archaea were targeted using high throughput 16S rRNA gene pyrosequencing analyses. A unique aspect of this study was the use of a universal set of 16S rRNA gene primers to characterize total and diffuse flow-specific microbial communities from varied deep-sea hydrothermal environments. Both surrounding seawater and diffuse flow water samples contained large numbers of Marine Group I (MGI) Thaumarchaea and Gammaproteobacteria taxa previously observed in deep-sea systems. However, these taxa were geographically distinct and segregated according to type of spreading center. Diffuse flow microbial community profiles were highly differentiated. In particular, EPR dominant diffuse flow taxa were most closely associated with chemolithoautotrophs, and off axis water was dominated by heterotrophic-related taxa, whereas the opposite was true for Guaymas Basin. The diversity and richness of diffuse flow-specific microbial communities were strongly correlated to the relative abundance of Epsilonproteobacteria, proximity to macrofauna, and hydrothermal system type. Archaeal diversity was higher than or equivalent to bacterial diversity in about one third of the samples. Most diffuse flow-specific communities were dominated by OTUs associated with Epsilonproteobacteria, but many of the Guaymas Basin diffuse flow samples were dominated by either OTUs within the Planctomycetes or hyperthermophilic Archaea. This study emphasizes the unique microbial communities associated with geochemically and geographically distinct hydrothermal diffuse flow environments.