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Growing season temperatures play a crucial role in controlling treeline elevation at regional to global scales. However, understanding of treeline dynamics in response to long-term changes in temperature is limited. In this study, we analyze pollen, plant macrofossils, and charcoal preserved in organic layers within a 10,400-year-old ice patch and in sediment from a 6000-year-old wetland located above present-day treeline in the Beartooth Mountains, Wyoming, to explore the relationship between Holocene climate variability and shifts in treeline elevation. Pollen data indicate a lower-than-present treeline between 9000 and 6200 cal yr BP during the warm, dry summer and cold winter conditions of the early Holocene. Increases in arboreal pollen at 6200 cal yr BP suggest an upslope treeline expansion when summers became cooler and wetter. A possible hiatus in the wetland record at ca. 4200–3000 cal yr BP suggests increased snow and ice cover at high elevations and a lowering of treeline. Treeline position continued to fluctuate with growing season warming and cooling during the late-Holocene. Periods of high fire activity correspond with times of increased woody cover at high elevations. The two records indicate that climate was an important driver of vegetation and treeline change during the Holocene. Early Holocene treeline was governed by moisture limitations, whereas late-Holocene treeline was sensitive to increases in growing season temperatures. Climate projections for the region suggest warmer temperatures could decrease effective growing season moisture at high elevations resulting in a reduction of treeline elevation.