Abstract Nitrification, a key process in the nitrogen cycle, involves the oxidation of ammonia to nitrite and nitrate by a diverse group of chemolithoautotrophic microorganisms. The order Nitrospirales (referred to in literature as the genus Nitrospira), which includes both nitrite-oxidizing and complete ammonia-oxidizing bacteria, plays a central role in this process. We sequenced the genomes of nine Nitrospirales members, incorporating genomes from previously unsequenced taxonomic Nitrospirales lineages. A comprehensive genomic analysis of these new Nitrospirales was conducted, which included an examination of their habitat distribution, phylogenetic diversity, and functional capabilities. This was complemented by the construction of and comparison to a database of 446 non-redundant, high-quality Nitrospirales genomes. Our phylogenomic analysis uncovered the presence of additional unclassified lineages and provided a comparison between genome-based and 16S rRNA gene-based taxonomies. Whereas some Nitrospirales lineages seem to exhibit habitat preferences, others are found across a wide variety of ecosystems, suggesting a broad niche spectrum. This capacity to adapt to different environmental conditions is also reflected in the high variability and modularity of the respiratory chain and nitrogen assimilation mechanisms. Additionally, we found evidence of quorum sensing systems in species beyond lineage II, implying a broader ecological role for this communication mechanism within the Nitrospirales. Finally, we identified a set of conserved genes unique to nitrite oxidoreductase-containing Nitrospirales, providing insights into the emergence of this functional group. In conclusion, our study emphasizes the adaptability of the various nitrifying classes of the order Nitrospirales to diverse environments and reveals the presence of new taxonomic lineages.