<p>Microbial safety is fundamental to ensuring water quality, particularly in the Yangtze River Basin, China’s most critical drinking water source. Despite its ecological and economic importance, the basin faces significant anthropogenic pressures, including wastewater discharge, which may elevate the risk of pathogenic contamination. However, fragmented sampling efforts and limited coverage have hindered a systematic understanding of pathogenic microbial diversity and distribution across this vast ecosystem. A novel bioinformatic pipeline leveraging Genome-Specific Markers to accurately identify and quantify potential pathogenic taxa in metagenomic data was applied to 625 publicly available metagenomes, spanning water, sediments, and riparian soils along the 6,300 km Yangtze River continuum. We reconstructed a potential pathogen catalog comprising 403 taxa, largely expanding the pathogen diversity in the large river ecosystem. We also generate the Richness distribution maps of potential pathogens for water, sediments and soils along Yangtze River. The basin-scale pathogen inventory not only establishes a baseline for potential pathogenic bacteria communities in the Yangtze Basin but also serves as a reference library for quick biosurveillance and risk management from genomic resolution.</p>