20Nitrite-oxidizing bacteria (NOB) play a critical role in the mitigation of nitrogen pollution from 21 freshwater systems by metabolizing nitrite to nitrate, which is removed via assimilation, 22 denitrification, or anammox. Recent studies revealed that NOB are phylogenetically and 23 metabolically diverse, yet most of our knowledge of NOB comes from only a few cultured 24 representatives. Using enrichment methods and genomic sequencing, we identified four novel 25 Candidatus Nitrotoga NOB species from freshwater sediments and water column samples in 26 Colorado, USA. Genome assembly revealed highly conserved 16S rRNA gene sequences, but a 27 surprisingly broad metabolic potential including genes for nitrogen, sulfur, hydrogen, and 28 organic carbon metabolism. Genomic predictions suggest that Nitrotoga can metabolize in low 29 oxygen or anaerobic conditions, which may support a previously unrecognized environmental 30 niche. An array of antibiotic and metal resistance genes likely allows Nitrotoga to withstand 31 environmental pressures in impacted systems. Phylogenetic analyses reveal a deeply divergent 32 nitrite oxidoreductase alpha subunit (NxrA) not represented in any other NOB, suggesting a 33 novel evolutionary trajectory for Nitrotoga. Nitrotoga-like 16S rRNA gene sequences were 34 prevalent in globally distributed environments. This work considerably expands our knowledge 35 of the Nitrotoga genus and improves our understanding of their role in the global nitrogen cycle.36 37