Lake of the Woods is one of North America’s largest lake complexes, covering an area of ∼4000 km2 in Ontario, Manitoba, and Minnesota. Multiple proxies and 38 new accelerator mass spectroscopy (AMS) radiocarbon dates from six sediment cores in the basin were used to interpret the Holocene paleo-environmental history of Lake of the Woods, including the late history of glacial Lake Agassiz. Sediments cored consist mainly of lacustrine silty clay to clayey silt. A distinct pink lamina in one core is associated with overflow into Lake Agassiz from the Superior basin related to the Marquette readvance of the Laurentide Ice Sheet 11,300 cal (9900 14C) yr B.P. Pedogenic zones developed in the sediment reflect periods when the lake floor was dry and exposed to weathering, specifically (1) during an initial phase after the Lake of the Woods basin became isolated from Lake Agassiz ca. 10,500–10,000 cal yr B.P. but before differential isostatic rebound caused water in the northern end of the basin to transgress south over the exposed lake floor, and (2) during a 3600-yr-long negative hydrological budget and chronological hiatus related to the mid-Holocene Hypsithermal warming and drying event, which is represented by <1 m of sediment. Diatoms first appear in the record after the Lake of the Woods became separated from Lake Agassiz shortly after 10,500 cal yr B.P. Ostracodes are present in sediments as young as ca. 7600 cal yr B.P., disappearing above the uppermost pedogenic zone; thecamoebians first appear after this. Distinct shifts in diatom assemblage composition and in sedimentary chlorophyll-a in one core track late Holocene changes in Lake of the Woods.