The presence, diversity, and distribution of a key group of subseafloor archaea, the Thermococcales, was examined in multiple diffuse flow hydrothermal vents at Axial Seamount, an active deep-sea volcano located in the northeast Pacific Ocean. A polymerase chain reaction (PCR) approach was used to determine if this group of subseafloor indicator organisms showed any phylogenetic distribution that may indicate distinct subseafloor communities at vents with different physical and chemical characteristics. Targeted primers for the Thermococcales 16S rRNA (small subunit ribosomal RNA) gene and intergenic transcribed spacer (ITS) region were designed and applied to organisms filtered directly from a variety of diffuse flow vents. Thermococcales were amplified from 9 of 11 samples examined, and it was determined that the ITS region is a better phylogenetic marker than the 16S rRNA in defining consistent groups of closely related sequences. Results show a relationship between environmental clone distribution and source vent chemistry. The most highly diluted vents with elevated iron and alkalinity contained a distinct group of Thermococcales as defined by the ITS region, suggesting separate subseafloor Thermococcales populations at diffuse vents within the Axial caldera.