Macroecological processes impact Australian soil resistomes and climatically stable regions with anthropogenic activities serve as ARG hotspots.

Soil antibiotic resistance genes (ARGs) pose a global health threat, but a critical knowledge gap remains regarding how macro-scale pedoclimatic constraints interact with land-use intensification to determine the spatial distribution of the soil resistome. To address this, we conducted a continental-scale survey of Australian topsoils and used metagenomic analysis to reveal the hierarchy of drivers shaping soil resistome. Machine learning was applied to predict the spatial ARG distribution across Australia. We found that, at the continental scale, climatic variability acts as the dominant filter on ARG distribution, overriding local soil properties and human disturbance. Unexpectedly, climatically stable regions, characterized by sandy and low-carbon soils in Southwestern Australia, emerged as ARG hotspots. We also demonstrated that anthropogenic land use amplifies ARG abundance within these climatically stable regions. Furthermore, spatial modelling revealed distinct geographical patterns: although total ARG abundance was enriched in coastal regions, specific resistance mechanisms showed unique distributions. As a continental-scale investigation of soil ARGs in Australia, this study provides a framework to identify high-risk regions where lower climatic variability and intensive farming interact to enhance antimicrobial resistance.