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AbstractComammox (CMX) Nitrospira bacteria (NB) can accomplish ammonia oxidation independently, and their niche differentiation holds promise for their ecological and survival functions. In this work, the vertical niche differentiation of CMX NB was investigated in the soils of 6 water-level fluctuation (WLF) zones (both natural and artificial) in the Three Gorges Reservoir (TGR) region. The results demonstrated that the level of clade A amoA was obviously reduced with increasing soil depth in the natural WLF zones and one of the artificial WLF zones. However, in the other two artificial WLF zones, the abundance of this gene was not dramatically reduced with depth. The level of clade B amoA did not markedly decrease with increasing soil depth in most WLF zones and remained stable in the three WLF zones. Total nitrogen (TN) had the most significant effect on the abundance of CMX NB. Clade A.1, clade A.2.1, clade A.2.2, clade A.3, and clade B of CMX NB co-occurred simultaneously in all WLF zones. The number of operational taxonomic units (OTUs) of clade A in the two types of WLF zones first increased and then decreased with increasing depth, whereas the number of OTUs of clade B continuously increased with depth in the artificial WLF zone. Total carbon (TC) and pH, as environmental factors, affected the community structure of CMX NB. This study confirmed the vertical differentiation of the abundance and diversity of CMX NB in the WLF zones of the TGR region, and the artificial restoration of the WLF zones affected the niche differentiation of CMX NB to a certain degree.