Abstract 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, extracted from sediments directly overlying surface-breaching gas hydrate mounds collected from a research submersible (water depth 550-575 m), were amplified with nine different 16S rDNA gene primer sets. The polymerase chain reaction primers targeted microorganisms at the domain-specific (Bacteria and Archaea) and group-specific (sulfate-reducing bacteria (SRB) and putative anaerobic methane-oxidizing (ANME) archaea) level. Amplicons were obtained with five of the nine primer sets including two of the six SRB Groups (SRB Group 5 and Group 6) and used to generate five different clone libraries. Analysis of 126 clones from the Archaea library revealed that the sediments associated with naturally occurring gas hydrate harbored a low diversity. Sequence analysis indicated the majority of archaeal clones were most closely related to Methanosarcinales, Methanomicrobiales and distinct phylogenetic lineages within the ANME groups. The most frequently recovered phylotypes in the ANME library were related to either ANME-2 or Methanomicrobiales. In contrast to the two archaeal libraries, bacterial diversity was higher with the majority of the 126 bacterial clones most closely related to uncultured clones dominated by the delta- and epsilon-Proteobacteria. Interestingly, while 82% of the clones in the SRB Group 5 library were affiliated with delta-Proteobacteria, the vast majority (83%) of clones in the SRB Group 6 library was affiliated with the Firmicutes. This is the first phylogenetic-based description of microbial communities extant in methane-rich hydrate-associated sediments from a hydrocarbon seep region in the Gulf of Mexico.