Abstract: While alleviating water scarcity, water diversion projects may also serve as important pathways for the cross-basin transport of per-and polyfluoroalkyl substances (PFASs). However, the occurrence and distribution of PFASs in the Wangyu River, the primary conveyance channel for the Yangtze-to-Taihu Water Diversion Project, remain poorly understood. Taking the Wangyu River as the research object, this study systematically investigated the contamination characteristics of 23 PFASs in the water body, adopted Spearman correlation analysis and principal component analysis to explore their potential sources, and carried out ecological and health risk assessments. The results showed that: ① Significant spatial differences in ΣPFASs concentrations were observed. Influenced by point-source emissions from the industrial parks, concentrations displayed a declining gradient from the upstream industrial parks to the downstream inlet of Taihu Lake. Specifically, the upstream industrial zone section was dominated by the novel alternative hexafluoropropylene oxide dimer acid (HFPO-DA) and short-chain perfluorohexanoic acid (PFHxA), while the middle and downstream regions were dominated by perfluorobutyric acid (PFBA) and perfluorooctanoic acid (PFOA) from the traditional perfluorocarboxylic acids.② Correlation and principal component analysis revealed that the characteristic pollutants (HFPO-DA, PFHxA) in the industrial park section were significantly separated from those in the middle and downstream regions (the cumulative variance contribution rate of the first two principal components reached 90.22%). This indicated different pollution sources and suggested that the PFASs in the industrial parks did not cause direct significant pollution to Taihu Lake through the Wangyu River during non-water-diversion periods.③ The multidimensional risk assessment found that, except for PFOA and PFOS, which posed a moderate risk to algae and fish, the risk quotient (RQ) values of the other five PFASs were all below 0.01, indicating acceptable risk levels.④ The health exposure assessment showed that there was no health risk of PFASs in drinking water intake of adults and children in the inlet section of Taihu Lake. However, the estimated daily intake (EDI) via drinking water route for children (11.25~11.36 ng/(kg·d)) was higher than that for adults (8.32~8.39 ng/(kg·d)). Future efforts should strengthen the continuous monitoring of PFASs and assess the risk of exposure to PFASs in the population through multiple pathways.