Per- and polyfluoroalkyl substances (PFAS) are pervasive environmental contaminants that pose significant threats to ecosystem health at regional and global scales. Despite their persistence, bioaccumulation potential, and toxicity (PBT), much remains unknown about their ecosystem-level effects in aquatic environments. Deciphering the “PFAS cycle” requires a detailed understanding of PFAS pools and fluxes across environmental compartments. Here, we present findings on PFAS partitioning among sediment, water, and biota in Great Lakes tributaries, revealing spatial contamination gradients and the roles of environmental media in PFAS dispersion. The Laurentian Great Lakes, the largest freshwater system in North America, supply drinking water to 40 million people, support immense biodiversity, and contribute significantly to regional economies, underscoring the critical importance of understanding PFAS dynamics in these systems. We used targeted PFAS analyses (LC-MS/MS) and adsorbable organic fluorine quantification (via PIGE) to assess complex PFAS profiles in northwestern Indiana (USA) watersheds draining into southern Lake Michigan. We found a gradient of shifting PFAS profiles likely influenced by industrialization and land-use patterns. In the majority of (77%) sampling sites in less developed watersheds, ΣPFAS levels were higher in fall compared to spring (0.266 compared to 0.065 ng/L), likely driven by reduced streamflow enhancing PFAS inputs. Conversely, sites in watersheds with more impervious surfaces exhibited elevated ΣPFAS in spring sampling (0.311 compared to 0.086 ng/L), potentially due to runoff that either disrupts seasonal flow patterns or contributes directly to PFAS loads. Analyses indicate that sediment is a significant reservoir for one dominant analyte, PFOS, which comprised 20%–70% of the PFAS profile at some sites. Additionally, sediment at sites within the Grand Calumet watershed displayed distinct signatures of polyfluoroalkyl phosphate esters (PAPs), precursor compounds commonly used as PFAS replacements in consumer products. These degrade into other toxic PFAS, highlighting the cycling and transformations of PFAS in the environment.