Thermoproteales populations (phylum Crenarchaeota) are abundant in high-temperature (>70° C) environments of Yellowstone National Park (YNP) and are important in mediating biogeochemical cycles of sulfur, arsenic and carbon. The objectives of this study were to determine specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph’s Coat Hot Spring [JCHS]; 80 °C; pH 6.1), and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as electron acceptors. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58 % G+C) and revealed numerous metabolic pathways for the degradation of carbohydrates, amino acids and lipids. Multiple dimethylsulfoxide molybdopterin (DMSO-MPT) oxidoreductase genes were identified, which are implicated in the reduction of sulfur and arsenic, as well as pathways for the de novo synthesis of nearly all required cofactors and metabolites. Comparative genomics of P. yellowstonensis versus assembled metagenome sequence from JCHS showed that this organism is highly-related (~95% average nucleotide identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.