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 PFASs associated with dissolved organic matter (DOM) is scarce, especially there is a lack of research on the effects of DOM with different molecular weights on the occurrence form of PFASs. To clarify the effects of DOM with different molecular weights on the occurrence form of PFASs in overlying water of rivers, the concentrations and composition of eleven PFASs (C4-C12) in overlying water of the Yangtze River were analyzed and the occurrence characteristics of PFASs associated with different molecular weight DOM were explored. The results showed that: (1) 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 PFASs in Wuhan were significantly higher than those in other sampling sites. (2) The concentration of DOM in the overlying water of the Yangtze River ranged from 0.08 mg/L to 3.84 mg/L. DOM was separated into <1 kDa, 1-3 kDa, 3-5 kDa, 5-10 kDa and >10 kDa by molecular weight. The concentration (1.56-3.84 mg/L) of dissolved organic carbon (DOC) of <1 kDa was significantly higher than that of other molecular weights. (3) For the detected PFASs, the concentration of PFASs associated with <1 kDa DOM (including freely dissolved PFASs) was significantly higher than that associated with other molecular weights DOM, accounting 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. This study shows that there are differences in the occurrence characteristics of PFASs associated with different molecular weight DOM in overlying water. Therefore, the concentration and bioavailability of PFASs associated with different molecular weight DOM should be considered comprehensively when conducting ecological risk assessment for PFASs in overlying water.