Per- and polyfluoroalkyl substances, or PFAS, gained significant public and regulatory attention due to widespread contamination and health harms associated with exposure. Ingestion of PFAS from contaminated food and water results in the accumulation of PFAS in the body and is considered a key route of human exposure. Here we calculate the potential contribution of PFOS from consumption of locally caught freshwater fish to serum levels. We analyzed data for over 500 composite samples of fish fillets collected across the United States from 2013 to 2015 under the U.S. EPA’s monitoring programs, the National Rivers and Streams Assessment and the Great Lakes Human Health Fish Fillet Tissue Study. The two datasets indicate that an individual’s consumption of freshwater fish is potentially a significant source of exposure to perfluorinated compounds. The median level of total targeted PFAS in fish fillets from rivers and streams across the United States was 9500 ng/kg, with a median level of 11,800 ng/kg in the Great Lakes. PFOS was the largest contributor to total PFAS levels, averaging 74% of the total. The median levels of total detected PFAS in freshwater fish across the United States were 278 times higher than levels in commercially relevant fish tested by the U.S. Food and Drug Administration in 2019–2022. Exposure assessment suggests that a single serving of freshwater fish per year with the median level of PFAS as detected by the U.S. EPA monitoring programs translates into a significant increase of PFOS levels in blood serum. The exposure to chemical pollutants in freshwater fish across the United States is a case of environmental injustice that especially affects communities that depend on fishing for sustenance and for traditional cultural practices. Identifying and reducing sources of PFAS exposure is an urgent public health priority.