Permafrost deposits at Bol'shoy Lyakhovsky Island are natural archives dating back to the Eemian (Krest Yuryakh Suite, ~125 kyr BP) with excellent conditions to preserve ancient DNA. The today treeless island is located between the Laptev and East Siberian Seas, but during the last glacial it was part of the Western Beringian landmass due to the marine regression, which exposed the shallow Siberian shelf. Our aim was to reconstruct vegetation changes driven by the strong climatic oscillations from the last interglacial until today. Permafrost coring took place west of the Zimov’e River where four terrestrial permafrost cores were drilled. In total we collected 72 sediment samples and combined pollen analysis with sedimentary ancient DNA metabarcoding for which we amplified the short vascular plant specific P6-loop of the plastid trnL (UAA) intron. Furthermore, we applied two newly developed larch-specific chloroplast SNP markers to assess their suitability in identifying past population dynamics from environmental samples. The new markers were re-sequenced and displayed both SNP variants of each marker in last interglacial samples. Highest diversity and a vegetation containing trees (Picea, Larix, Populus) were inferred covering the island during the last interglacial. During the Mid Weichselian interstadial and the Bølling-Allerød interstadial complex only Larix was detected among trees but disappeared along with most shrub taxa during the Holocene. This suggests that the northern extent of the Siberian treeline was further north than previously inferred from pollen analyses, and that modern larch taiga is differently composed than the taiga during the last interglacial.