Abstract: At present, studies on the distribution of polyfluoroalkyl substances (PFASs) in the aquatic environment mainly focus on their total dissolved concentration, while the studies about polyfluoroalkyl substances associated with dissolved organic matter (DOM) is scarce, especially the distribution characteristics of polyfluoroalkyl substances associated with different molecular weight DOM are ignored. In this study, the concentrations and composition of eleven PFASs (C4-C12) in overlying water of the Yangtze river were analyzed and the distribution characteristics of PFASs associated with different molecular weight DOM were explored. The results showed that the average total dissolved concentration of PFASs in the overlying water of the Yangtze River was 52.6 ng/L, and perfluoropentanoic acid (PFPeA) and perfluorohexanoic acid (PFHxA) were the main monomer pollutants. Due to the influence of point source pollution, the total dissolved concentration and monomer concentration of polyfluoroalkyl substances in Wuhan were significantly higher than those in other sampling sites. The concentration of DOM in the overlying water of the Yangtze River ranged from 0.08 mg/L to 3.84 mgC/L. DOM was separated into <1 kDa, 1-3 kDa, 3-5 kDa, 5-10 kDa and >10 kDa by molecular weight. The concentration of dissolved organic carbon (DOC, 1.56-3.84 mg/L) of <1 kDa was significantly higher than that of other molecular weights. For the detected polyfluoroalkyl substances, the concentration of PFASs associated with <1 kDa DOM (including freely dissolved PFASs) was significantly higher than that of PFASs associated with other molecular weights DOM, and accounted for more than 85% of the total dissolved PFASs in overlying water, indicating that the PFASs associated with DOM in overlying water had high bioavailability.