Boreal regions and their freshwater ecosystems may be susceptible to future climate change under projected warmer conditions. Northwest Ontario is a boreal region adjacent to the climatically sensitive prairie-forest ecotone (PFE). Pollen records spanning the Holocene from near the Manitoba/Ontario border to lakes up to ~300 km east of the PFE indicate a warmer and possibly wetter mid-Holocene period across northwest Ontario from ~8000 to 4500 cal. yr BP. To date, only one Holocene-scale record of changes in effective moisture, as indicated through diatom-inferred changes in lake level (Experimental Lakes Area (ELA) Lake 239), is available from this region. Our study expands the regional context of Holocene climate changes, with the analysis of diatom assemblages in sediment cores from two additional lakes, which span a distance of over 200 km across the present-day boreal forest, from 80 km west of ELA Lake 239 to ~150 km to the northeast. In cores from both lakes, benthic taxa predominate in the early-to-mid-Holocene period, with a low abundance of planktonic taxa, suggesting lower lake levels by ~2–5 m. Increases in the abundance of planktonic taxa to >50% occurred in both lakes beginning ~4500–4000 cal. yr BP suggesting positive water balance over the last 4000 years in comparison with the mid-Holocene period. This new evidence supports a regional mid-Holocene period of aridity, with reduced water levels across the boreal region of northwest Ontario. If future climate change results in lower effective moisture, then conditions could become similar to the mid-Holocene period aridity, leading to real challenges for the management of water resources across the region. © The Author(s) 2015.