Nitrifying microorganisms occur across a wide temperature range from 4 to 84 °C and previous studies in geothermal systems revealed their activity under extreme conditions. Archaea were detected to be responsible for the first step of nitrification, but it is still a challenging issue to clarify the identity of heat-tolerant nitrite oxidizers. In a long-term cultivation approach, we inoculated mineral media containing ammonium and nitrite as substrates with biofilms and sediments of two hot springs in Yellowstone National Park (USA). The nitrifying consortia obtained at 70 °C consisted mostly of novel Chloroflexi as revealed by metagenomic sequencing. Among these, two deep-branching novel Chloroflexi were identified as putative nitrite-oxidizing bacteria (NOB) by the presence of nitrite oxidoreductase encoding genes in their genomes. Stoichiometric oxidation of nitrite to nitrate occurred under lithoautotrophic conditions, but was stimulated by organic matter. Both NOB candidates survived long periods of starvation and the more abundant one formed miniaturized cells and was heat resistant. This detection of novel thermophilic NOB exemplifies our still incomplete knowledge of nitrification, and indicates that nitrite oxidation might be an ancient and wide-spread form of energy conservation.