Concentrations of Total40 per- and polyfluoroalkyl substances (PFAS), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), and Total13 PFAS were consistently higher at the AFFF sites compared to the two East Coast reference sites in tree swallow eggs, nestlings, and diet by as much as a factor of 40 times higher. Sites in the Upper Midwest with other PFAS sources also had substantial, but qualitatively lower concentrations of PFAS compared to the AFFF-source sites. Perfluorooctane sulfonate was the only PFAS detected in all samples in all matrices. Concentrations of most other PFAS, such as the carboxylates and fluorotelomers among other PFAS, did not differ between AFFF and reference sites indicating that most other PFAS were not a significant proportion of the foam formulations used at these DOD sites and that they entered the environment from other sources. Perhaps indicating that diet is the dominant vector of exposure to tree swallows, there was a high correlation between concentration of Total40 in the diet (flying insects) and concentrations in both eggs (r = 0.82) and nestling carcasses (r = 0.93). Perfluorohexane sulfonate, the second most common constituent of the legacy AFFF formulations, was <1% of the Total40 at the reference sites in both eggs and nestlings, but PFHxS comprised up to 9.7% (eggs) and 9.0% (nestlings) at AFFF-influenced sites so it may be a good indicator PFAS to identify historical AFFF sources. The daily probability of both egg and nestling survival was similar across all sites and there was no association between the probability of daily egg or nestling survival and Total40 PFAS. There was also no association between these two measures of reproductive success and concentrations of individual PFAS. Two immune response metrics (haptoglobin-like activity [PIT54] and total immunoglobulin Y [IGY]) did not differ among sites. There were few differences among sites for the other biomarker assessments nor correlations with PFAS concentrations.