North American river otters (Lontra canadensis) aretop predators in riverine ecosystems and are vulnerable to per- andpolyfluoroalkyl substance (PFAS) exposure. Little is known aboutthe magnitude of exposure and tissue distribution of PFAS in riverotters. We measured 45 PFAS in various tissues of 42 river otterscollected from several watersheds in the state of West Virginia,USA. The median concentrations of ΣAll (sum concentration of45 PFAS) varied among tissues in the following decreasing order:liver (931 ng/g wet weight) > bile > pancreas > lung > kidney >blood > brain > muscle. Perfluoroalkylsulfonates (PFSAs) were thepredominant compounds accounting for 58−75% of the totalconcentrations, followed by perfluoroalkyl carboxylates (PFCAs;21−35%). 8:2 fluorotelomer sulfonate (8:2 FTS), 10:2 FTS, and 6:2 chlorinated polyfluoroalkyl ether sulfonate were frequentlyfound in the liver (50−90%) and bile (96−100%), whereas hexafluoropropylene oxide dimer acid (HFPO-DA) was rarely found.The hepatic concentrations of ΣAll in river otters collected downstream of a fluoropolymer production facility located along theOhio River were 2-fold higher than those in other watersheds. The median whole body burden of ΣAll was calculated to be 1580μg. PFOS and perfluorooctanoic acid (PFOA) concentrations in whole blood of some river otters exceeded the human toxicityreference values, which warrant further studies.