Chemoautotrophic canonical ammonia oxidizers (ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB)) and complete ammonia oxidizers (comammox Nitrospira) are accountable for ammonia oxidation, which is a fundamental process of nitrification in terrestrial ecosystems. However, the relationship between autotrophic nitrification and the active nitrifying populations during N-urea incubation has not been totally clarified. The N-labeled DNA stable isotope probing (DNA-SIP) technique was utilized in order to study the response from the soil nitrification process and the active nitrifying populations, in both acidic and neutral paddy soils, to the application of urea. The presence of CH almost completely inhibited NO-N production, indicating that autotrophic ammonia oxidation was dominant in both paddy soils. N-DNA-SIP technology could effectively distinguish active nitrifying populations in both soils. The active ammonia oxidation groups in both soils were significantly different, AOA (NS (Nitrososphaerales)-Alpha, NS-Gamma, NS-Beta, NS-Delta, NS-Zeta and NT (Ca. Nitrosotaleales)-Alpha), and AOB (Nitrosospira) were functionally active in the acidic paddy soil, whereas comammox Nitrospira clade A and Nitrosospira AOB were functionally active in the neutral paddy soil. This study highlights the effective discriminative effect of N-DNA-SIP and niche differentiation of nitrifying populations in these paddy soils. KEY POINTS: • N-DNA-SIP technology could effectively distinguish active ammonia oxidizers. • Comammox Nitrospira clade A plays a lesser role than canonical ammonia oxidizers. • The active groups in the acidic and neutral paddy soils were significantly different.