Summer warming is driving a greening trend across the Arctic, with the potential for large-scale amplification of climate change due to vegetation-related feedbacks [Pearson et al., Nat. Clim. Chang. (3), 673–677 (2013)]. Because observational records are sparse and temporally limited, past episodes of Arctic warming can help elucidate the magnitude of vegetation response to temperature change. The Last Interglacial ([LIG], 129,000 to 116,000 y ago) was the most recent episode of Arctic warming on par with predicted 21st century temperature change [Otto-Bliesner et al., Philos. Trans. A Math. Phys. Eng. Sci. (371), 20130097 (2013) and Post et al., Sci. Adv. (5), eaaw9883 (2019)]. However, high-latitude terrestrial records from this period are rare, so LIG vegetation distributions are incompletely known. Pollen-based vegetation reconstructions can be biased by long-distance pollen transport, further obscuring the paleoenvironmental record. Here, we present a LIG vegetation record based on ancient DNA in lake sediment and compare it with fossil pollen. Comprehensive plant community reconstructions through the last and current interglacial (the Holocene) on Baffin Island, Arctic Canada, reveal coherent climate-driven community shifts across both interglacials. Peak LIG warmth featured a ∼400-km northward range shift of dwarf birch, a key woody shrub that is again expanding northward. Greening of the High Arctic—documented here by multiple proxies—likely represented a strong positive feedback on high-latitude LIG warming. Authenticated ancient DNA from this lake sediment also extends the useful preservation window for the technique and highlights the utility of combining traditional and molecular approaches for gleaning paleoenvironmental insights to better anticipate a warmer future. Significance The Arctic is warming exceptionally rapidly, promoting an expansion of shrubs across the Arctic with global-scale climate implications. The Last Interglacial (∼125,000 y ago) was the most recent time the Arctic was warmer than present and thus serves as an analogue for Arctic greening in the near future. Ancient plant DNA in lake sediment from this time reveals major ecosystem changes in response to warmth, including an ∼400 km northward shift of dwarf birch relative to today. Enhanced shrub cover, corroborated by molecular and microfossil analyses, amplified warming during the Last Interglacial and will likely play a similar role in the future. This record constitutes the oldest authenticated plant DNA from lake sediment yet reported, increasing the technique’s temporal potential.