Per- and polyfluoroalkyl substances (PFAS) have become an environmental issue worldwide. A first step to assessing potential adverse effects on fish populations is to determine if concentrations of concern are present in a region and if so, in which watersheds. Hence, plasma from adult smallmouth bass Micropterus dolomieu collected at 10 sites within 4 river systems in the mid-Atlantic region of the United States, from 2014 to 2019, was analyzed for 13 PFAS. These analyses were directed at better understanding the presence and associations with land use attributes in an important sportfish. Four substances, PFOS, PFDA, PFUnA, and PFDoA, were detected in every plasma sample, with PFOS having the highest concentrations. Sites with mean plasma concentrations of PFOS below 100 ng/ml had the lowest percentage of developed landcover in the upstream catchments. Sites with moderate plasma concentrations (mean PFOS concentrations between 220 and 240 ng/ml) had low (< 7.0) percentages of developed land use but high (> 30) percentages of agricultural land use. Sites with mean plasma concentrations of PFOS > 350 ng/ml had the highest percentage of developed land use and the highest number PFAS facilities that included military installations and airports. Four of the sites were part of a long-term monitoring project, and PFAS concentrations of samples collected in spring 2017, 2018, and 2019 were compared. Significant annual differences in plasma concentrations were noted that may relate to sources and climatic factors. Samples were also collected at two sites for tissue (plasma, whole blood, liver, gonad, muscle) distribution analyses with an expanded analyte list of 28 PFAS. Relative tissue distributions were not consistent even within one species of similar ages. Although the long-chained legacy PFAS were generally detected more frequently and at higher concentrations, emerging compounds such as 6:2 FTS and GEN X were detected in a variety of tissues.