DNA-based snapshots of ancient vegetation have shown that the composition of high-latitude plant communities changed considerably during the late Quaternary. However, parallel changes in biotic interactions remain largely uninvestigated. Here we show how mutualisms involving plants and heterotrophic organisms varied during the last 50,000 years. During 50-25 ka BP, a cool period featuring stadial-interstadial fluctuations, arbuscular mycorrhizal and non-N-fixing plants predominated. During 25-15 ka BP, a cold, dry interval, the representation of ectomycorrhizal, non-mycorrhizal and facultatively mycorrhizal plants increased, while that of N-fixing plants decreased further. From 15 ka BP, which marks the transition to and establishment of the Holocene interglaciation, representation of arbuscular mycorrhizal plants decreased further, while that of ectomycorrhizal, non-mycorrhizal, N-fixing and wind-pollinated plants increased. These changes in the mutualist trait structure of vegetation may reflect responses to historical environmental conditions that are without current analogue, or biogeographic processes, such as spatial decoupling of mutualist partners.