A molecular phylogenetic approach was used to characterize the composition of microbial communities from two gas hydrate sedimentary systems in the Gulf of Mexico. Nucleic acids were extracted from three distinct locales on surface breaching gas hydrate mounds, i.e., sediment overlaying gas hydrate, sediment/hydrate interface and sediment-free hydrate, and from three sediment depths, i.e., 0-2, 6-8 and 10-12 cm, in Beggiatoa sp. mat-associated sediments located several meters from exposed gas hydrate. Samples were collected from a research submersible (water depth 550-575 m) during two research cruises aboard the R/V Seward Johnson I and II funded by the NSF Life in Extreme Environments program. The 16S rRNA gene and 16S rRNA were amplified using PCR and reverse transcription-PCR, respectively, from DNA and RNA extracted from the total microbial community. The primers targeted microorganisms at the domain-specific, i.e., Bacteria and Archaea, and group-specific, i.e., sulfate-reducing bacteria (SRB) and putative anaerobic methane-oxidizing (ANME) archaea, level. Sequence analysis of the Bacteria clones revealed that the microbial communities were primarily dominated by Deltaproteobacteria. Other Proteobacteria classes, including Epsilon- and Gammaproteobacteria, represented a large fraction of the total microbial community isolated from the sediment overlying hydrate sample and the metabolically active fraction of the 0-2 cm sediment depth sampled from the Beggiatoa sp. mat-associated sediments. Sequence analysis indicated the majority of the archaeal clones were most closely related to Methanosarcinales, Methanomicrobiales and distinct lineages within the ANME groups. Several novel lineages were identified including a fourth ANME-2 clade, i.e., ANME-2D, and three clades with no closely related previously sequenced 16S rRNA gene clones or isolates, i.e., Unclassified Bacteria groups 1 and 2 and Unclassified Euryarchaeota. These studies represent the first 16S rRNA gene and 16S rRNA phylogenetic-based description of microbial communities extant in sediment-free gas hydrate and in methane-rich hydrate-associated and Beggiatoa sp.-associated sediments from a hydrocarbon seep region in the Gulf of Mexico.