Terminal-restriction fragment length polymorphisms (T-RFLPs) were used to address the question whether there are multiple phylotypes of bacteria within the trophosome of the vestimentiferan tubeworm Ridgeia piscesae in addition to the known endosymbiont. Clone libraries were constructed to aid in the identification of the additional bacterial phylotypes. Individual R. piscesae specimens were collected from Juan de Fuca Ridge, Axial Caldera and Explorer Ridge, Magic Mountain. Clone library analyses revealed only one bacterial phylotype (expected R. piscesae endosymbiont) within the Explorer Ridge trophosomes. However, the Axial Caldera clone library revealed five operational taxonomic units (OTUs). Three of the resulting phylotypes detected (designated RAE) were putative thioautotrophic symbionts within the γ-Proteobacteria which belonged to the anticipated endosymbiont group (RAE OTU 1), the Maorithyas hadalis symbiont II group (RAE OTU 3), and the Halothiobacillus group (RAE OTU 4). The remaining two phylotypes were most likely opportunistically derived. RAE OTU 5 was an α-Proteobacterium within the Roseobacter group and RAE OTU 2 was within the Cytophaga–Flavobacterium–Bacteroidetes. T-RFLP analyses revealed that all 15 trophosomes evaluated contained at least RAE OTUs 1 and 4. Five of the 15 trophosomes contained at least RAE OTUs 1, 3, and 4 and two of the trophosomes contained at least five RAE OTUs. Cluster analysis of the T-RFLP data revealed three distinct clusters. The number of taxa within the trophosome may be an indication of the general health of the tubeworm. This study strongly suggests that we have discovered and identified more than one bacterial phylotype within the trophosome of vestimentiferan R. piscesae.