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During recent decades, lake levels in the Yukon Flats region of interior Alaska have fluctuated dramatically. However, prior to recorded observations, no data are available to indicate if similar or more extreme variations occurred during past centuries and millennia. This study explores the history of Yukon Flats lake origins and lake levels for the past approximately 5,500 years from sediment analyses guided by previous work on permafrost extent, thermokarst, and modern isotope hydrology. Sediments dated by Pb-210 and AMS radiocarbon indicate stable chronologies following initial lake initiation. Subsequent lithology is autochthonous, and oxygen isotope ratios of endogenic carbonate reflect lake level change at multiple time scales. Sediment results indicate high lake levels between approximately 4000 and 1850 cal yr BP, which is interpreted to reflect wetter-than-modern conditions. Lower lake levels with short-lived high stands during the past approximately 800 years reflect generally arid conditions with brief wet intervals similar to the region’s moisture regime today. The millennial trend is one of increasing aridity and corresponds closely with fire reconstructions and regional paleoclimatic trends. We conclude that high-magnitude lake-level fluctuations and decadal scale trends occurred before the observational period and are persistent hydroclimatic features of the Yukon Flats region.