Per- and polyfluoroalkyl substances (PFAS) are a large and growing class of chemicals gaining global attention due to their persistence, mobility, and toxicity. Given the diverse chemical properties of PFAS and their varying distributions in water and tissue, monitoring of different matrices is critical to determine their presence and accumulation. Here, we used a platform combining liquid chromatography, ion mobility spectrometry, and high-resolution mass spectrometry for non-targeted analysis to detect and identify PFAS in alligator plasma from North Carolina (5 years, 2018-2022) and Florida (2021 only). Structures for 12 PFAS were elucidated, including 2 novel structures, and an additional 34 known PFAS were detected. Three of these compounds were previously unreported in environmental media. More PFAS were detected in North Carolina alligators and no novel PFAS were detected in Florida gators. Quantitative analysis of 21 of the known PFAS revealed that plasma concentrations did not change over the 5-year study.