The accelerating threat from antimicrobial resistance (AMR) has become a global health issue. The properties of AMR in the gut microbiome of preterm infants and its clinical relevance with necrotizing enterocolitis (NEC) remain unknown. In-depth integrative analyses of 5,684 gut metagenomes are performed to build an AMR genes (ARGs) landscape. A subset of 107 preterm infants who developed NEC is sampled to examine the trajectory and predictive potential of ARGs preceding NEC onset. The variation and core set of ARGs, their higher burden and diversity, and potential ARGs-enriched gut bacteria in preterm infants compared to full-term infants are comprehensively discovered, reflecting a strain shift in genomic functions. Moreover, the gut resistome converged over 9 days before NEC onset is observed, which is driven by 24 ARGs. Machine learning analysis reveals potential usage of the gut resistome as an indicator for predicting NEC onset in an external validation preterm birth cohort (the area under the receiver operating characteristic curve, AU-ROC = 0.823), which is significantly higher than that based on the bacterial species (AU-ROC = 0.727). Overall, the findings can be referenced to mitigate the burden and spread of ARGs, and specific ARGs have potential for disease risk stratification to improve clinical management.© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.