Pollen and diatom assemblages from well-dated sediment cores from three lakes forming a west-to-east transect across the boreal forest in northwest Ontario (Canada) were used to evaluate the timing and nature of the movement of the prairie-forest ecotone (PFE) across the Holocene. Changes in vegetation, temperature, and effective moisture were inferred from pollen and pollen-based transfer functions. Analyses indicated site-specific vegetational and climate changes across short spatial distances, with prolonged prairie-like conditions during the middle Holocene at the westernmost site. Increased reconstructed temperatures at this westernmost site occurred from ~9000 to 3000 cal yr BP, alongside increases in diatom-inferred lake levels beginning at ~6000 cal yr BP. The abundance of Quercus peaked concurrently with rising lake levels before declining to trace levels by ~3000 cal yr BP. Increases in the abundance of non-arboreal pollen between ~8500 and ~4500 cal yr BP at the more eastern lakes suggest relatively delayed and truncated PFE influence, before the reestablishment of primarily boreal taxa by ~4500 cal yr BP, coincident with diatom-inferred increases in lake levels. This study shows that the PFE moved both farther east and north than previously determined, but generally agrees with established patterns in vegetation from other studied regions along the PFE. Copyright © University of Washington. Published by Cambridge University Press, 2021.