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Abstract Subannual climate reconstructions of the Holocene are rare despite the ability of such records to provide a better understanding of the underlying factors that drive subannual climate variability. We used specialized confocal laser fluorescent microscope imaging and automated secondary ion mass spectrometry microanalysis to resolve a seasonal oxygen isotope (δ18O) record of a late Holocene–aged (2.7–2.1 ka) speleothem from mid-continental North America. We did this by measuring intra-band δ18O variability (Δ18O) within 117 annual bands over a 600 yr span of the late Holocene. We interpret a change in Δ18O values after 2.4 ± 0.1 ka to reflect an increase in the amount of winter precipitation. Our study produced direct measurements of past seasonality, offers new insights into shifting seasonal precipitation patterns that occurred during the late Holocene in central North America, and adds a new tool for understanding the complex precipitation and temperature histories of this region.