Ambient nitrous oxide (N2O) emissions from Great Boiling Spring (GBS) in the US Great Basin depended on temperature, with the highest flux, 67.8 ± 2.6 μmol N2O-N m−2 day−1, occurring in the large source pool at 82 °C. This rate of N2O production contrasted with negligible production from nearby soils and was similar to rates from soils and sediments impacted with agricultural fertilizers. To investigate the source of N2O, a variety of approaches were used to enrich and isolate heterotrophic micro-organisms, and isolates were screened for nitrate reduction ability. Nitrate-respiring isolates were identified by 16S rRNA gene sequencing as Thermus thermophilus (31 isolates) and T. oshimai (three isolates). All isolates reduced nitrate to N2O but not to dinitrogen and were unable to grow with N2O as a terminal electron acceptor. Representative T. thermophilus and T. oshimai strains contained genes with 96–98% and 93% DNA identity, respectively, to the nitrate reductase catalytic subunit gene (narG) of T. thermophilus HB8. These data implicate T. thermophilus and T. oshimai in high flux of N2O in GBS and raise questions about the genetic basis of the incomplete denitrification pathway in these organisms and on the fate of biogenic N2O in geothermal environments.