The current state of permafrost in Alaska and meaningful expectations for its future evolution are informed by long-term perspectives on previous permafrost degradation. Thermokarst processes in permafrost landscapes often lead to widespread lake formation and the spatial and temporal evolution of thermokarst lake landscapes reflects the combined effects of climate, ground conditions, vegetation, and fire. This study provides detailed analyses of thermokarst lake sediments of Holocene age from the southern loess uplands of the Yukon Flats, including bathymetry and sediment core analyses across a water depth transect. The sediment core results, dated by radiocarbon and 210Pb, indicate the permanent onset of finely laminated lacustrine sedimentation by ∼8,000 cal yr BP, which followed basin development through inferred thermokarst processes. Thermokarst expansion to modern shoreline configurations continued until ∼5000 cal yr BP and may have been influenced by increased fire. Between ∼5000 and 2000 cal yr BP, the preservation of fine laminations at intermediate and deep-water depths indicate higher lake levels than present. At that time, the lake likely overflowed into an over-deepened gully system that is no longer occupied by perennial streams. By ∼2000 cal yr BP, a shift to massive sedimentation at intermediate water depths indicates that lake levels lowered, which is interpreted to reflect a response to drier conditions based on correspondence with Yukon Flats regional fire and local paleoclimate reconstructions. Consideration of additional contributing mechanisms include the possible influence of catastrophic lake drainages on down-gradient base-flow levels that may have enhanced subsurface water loss, although this mechanism is untested. The overall consistency between the millennial lake-level trends documented here with regional paleoclimate trends indicates that after thermokarst lakes formed, their size and depth has been affected by North Pacific atmospheric circulation in addition to the evolution of permafrost, ground ice, and subsurface hydrology. As the first detailed study of a Holocene thermokarst basin that links expansion, stabilization and subsequent climate-driven lake level variations in a loess upland, these results provide a framework for future investigations of paleoclimatic signals from similar lake systems that characterize large regions of Alaska and Siberia.