Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with known bioaccumulative and toxic effects in aquatic ecosystems. This study assessed site-specific differences in PFAS contamination in fish from Ashumet Pond, Sudbury River, and Great Herring Pond (reference site) in Massachusetts. Fish from Ashumet Pond exhibited the highest PFAS concentrations, particularly perfluorooctane sulfonate (PFOS), which exceeded levels in plasma almost 650 times those at the reference site. Principal component analysis identified distinct PFAS profiles at each site, reflecting localized contamination sources. Temporal analysis at Ashumet Pond revealed a substantial increase in plasma PFOS and perfluorodecanoic acid (PFDA) from 2020 to 2022. Tissue distribution analyses showed the highest PFAS concentrations in plasma, followed by liver and muscle, consistent with PFAS binding affinity for blood proteins. Species-specific differences in PFAS bioaccumulation were observed, with largemouth bass (Micropterus nigricans) exhibiting higher body burdens than banded killifish (Diaphanus fundulus), likely due to trophic position and dietary exposure. Histopathological assessments and gene transcript analyses revealed associations between PFAS exposure and inflammatory responses, oxidative stress, endocrine disruption, and immune-related pathways, with the most pronounced molecular effects observed at the downstream site of the Sudbury River. This study underscores the importance of understanding site-specific contamination sources, exposure pathways, and biological effects of PFAS in fish. These findings would benefit from additional research on sediment contamination, temporal analyses at each site, trophic transfer, and transcriptomic analyses across multiple organs to further elucidate PFAS toxicity mechanisms and guide remediation efforts.