Background and aims <p>Traditionally, ammonia oxidation has been attributed to ammonia-oxidizing bacteria (AOB) and archaea (AOA). However, complete ammonia-oxidizing bacteria (Comammox) can perform nitrification in its entirity, transforming current understanding of microbial N cycling and necessitating further investigation. Here, the influence of different maize-soybean rotation systems on AOA, AOB, and Comammox community composition and abundance were examined in farmland soils under drip fertigation in semi-arid regions of Northeastern China.</p> Methods <p>Experimental treatments included soybean continuous cropping (SC) and three rotations: maize-soybean (MS), maize-maize-soybean (MMS), and maize-soybean-soybean (MSS). Quantitative fluorescence PCR and high-throughput sequencing were employed to assess microbial abundance and community structures.</p> Results <p>The present findings revealed that, soybean rotation enhanced hydroxylamine oxidase (HAO) and ammonia monooxygenase (AMO) activities, as well as soil nitrification potential (PNR). Among the rotation treatments, MMS and MSS exhibited significantly higher PNR and enzymatic activities than MS. AOA and AOB were significantly more abundant in rotation systems than in SC. However, in contrast, Comammox levels were markedly lower in rotation treatments. Community structure analysis revealed significant variation in AOA, AOB, and Comammox between rotation systems and SC, with organic matter (SOM), alkali hydrolyzed nitrogen (AN), and ammonium nitrogen (NH₄⁺-N) found to drive these shifts. Structural changes in AOA, AOB, and Comammox community compositions were found to directly influence PNR. Notably, relative Comammox contributions to nitrification surpassed that of conventional ammonia oxidizers.</p> Conclusions <p>In conclusion, these results suggest that Comammox contributed predominantly to ammonia oxidation in soybean continuous cropping and rotation systems under drip fertigation.</p> Graphical Abstract <p></p>