Per- and polyfluoroalkyl substances (PFAS) accumulate at the air-water interface of the surface microlayer (SML) on marine and freshwater bodies. In order to determine if including the SML when sampling bulk surface water leads to a high bias in measured PFAS concentrations, a pilot study and a full field study were conducted. The pilot study conducted at two sites was aimed at determining the analytical precision and small-scale (~1 m) spatial variability in concentrations of PFAS in bulk water and the SML. The full field study was performed at 11 sites, where three commonly used bulk surface water sampling methods were compared: (1) a peristaltic pump with tubing that excludes the SML, (2) a fully submerged sample bottle that excludes the SML, and (3) a partially submerged sample bottle that allows inclusion of the SML. The SML was sampled using the glass plate method. The samples were analyzed by liquid chromatography tandem mass spectrometry. The pilot study indicated that sampling variation was greater than analytical variation (although Levene's tests indicated that the differences were not statistically significant) and that relatively small differences in the mean concentration among sampling methods could be detected. The full investigation indicated that there was no evidence of high bias of PFAS concentrations in bulk surface water resulting from inclusion of SML using the partially submerged bottle sampling method. Unexpectedly, there was evidence that samples collected using the partially submerged bottle had slightly lower PFAS concentrations, particularly for less hydrophobic PFAS, than bulk water samples that excluded the SML. Additionally, the PFAS enrichment factor in the SML increased with increasing retention time, although the increase was not evident at all sampling sites for all PFAS. Integr Environ Assess Manag 2024;1-12. © 2024 SETAC.