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Purpose: Temperature is a key factor influencing the growth and activities of microorganisms and their associated biogeochemical processes including nitrification. However, limited studies have been performed to evaluate the response of the newly discovered comammox Nitrospira to temperature change and nitrogen (N) addition in agroecosystems, which has hindered our understanding of the ecophysiology and environmental adaption of comammox Nitrospira in agroecosystems. Materials and methods: We set up a microcosm experiment with 3 temperature treatments (15 °C, 25 °C, and 35 °C) × 3 levels of N addition (0 mg kg−1, 25 mg kg−1, and 50 mg kg−1) in an acidic Ultisol. We explored the impact of temperature and N addition on the abundance and community structure of comammox Nitrospira. Results and discussion: The addition of N and increasing temperature generally increased the net nitrification rate. The comammox Nitrospira clade A abundance decreased with the increase of temperature, indicating that clade A might favor environments with relatively lower temperature. However, the addition of N did not significantly influence the abundance of comammox Nitrospira clade A. Moreover, temperature had a stronger effect than N addition on the community structure of comammox Nitrospira. Clade A.2.1 were the exclusively dominant comammox Nitrospira phylotype in the tested soils, and were not affected by temperature and N addition, suggesting that temperature influenced comammox Nitrospira mainly through regulating the internal subclades of clade A.2.1. Conclusions: Our findings provide novel evidence that temperature had a stronger effect than N addition on the abundance and community structure of comammox Nitrospira and suggest that comammox Nitrospira are temperature sensitive and favor relatively lower temperature in the acidic Ultisols. [ABSTRACT FROM AUTHOR]