ABSTRACTThe symbiotic relationship between vestimentiferan tubeworms and their intracellular chemosynthetic bacteria is one of the more noteworthy examples of adaptation to deep-sea hydrothermal vent environments. The tubeworm symbionts have never been cultured in the laboratory. Nucleotide sequences from the small subunit rRNA gene suggest that the intracellular symbionts of the eastern Pacific vent tubewormsOasisia alvinae,Riftia pachyptila,Tevnia jerichonana, andRidgeia piscesaebelong to the same phylotype of gammaproteobacteria, “CandidatusEndoriftia persephone.” Comparisons of symbiont genomes between the East Pacific Rise tubewormsR. pachyptilaandT. jerichonanaconfirmed that these two hosts share the same symbionts. TwoRidgeiasymbiont genomes were assembled from trophosome metagenomes from worms collected from the Juan de Fuca Ridge (one and five individuals, respectively). We compared these assemblies to those of the sequencedRiftiaandTevniasymbionts. Pangenome composition, genome-wide comparisons of the nucleotide sequences, and pairwise comparisons of 2,313 orthologous genes indicated that “Ca. Endoriftia persephone” symbionts are structured on large geographical scales but also on smaller scales and possibly through host specificity.IMPORTANCERemarkably, the intracellular symbionts of four to six species of eastern Pacific vent tubeworms all belong to the same phylotype of gammaproteobacteria, “CandidatusEndoriftia persephone.” Understanding the structure, dynamism, and interconnectivity of “Ca. Endoriftia persephone” populations is important to advancing our knowledge of the ecology and evolution of their host worms, which are often keystone species in vent communities. In this paper, we present the first genomes for symbionts associated with the speciesR. piscesae, from the Juan de Fuca Ridge. We then combine these genomes with published symbiont genomes from the East Pacific Rise tubewormsR. pachyptilaandT. jerichonanato develop a portrait of the “Ca. Endoriftia persephone” pangenome and an initial outline of symbiont population structure in the different host species. Our study is the first to apply genome-wide comparisons of “Ca. Endoriftia persephone” assemblies in the context of population genetics and molecular evolution.