Arctic and alpine aquatic ecosystems are changing rapidly under recent global warming, threatening water resources by diminishing trophic status and changing biotic composition. Macrophytes play a key role in the ecology of freshwaters and we need to improve our understanding of long-term macrophytes diversity and environmental change so far limited by the sporadic presence of macrofossils in sediments. In our study, we applied metabarcoding using the trnL P6 loop marker to retrieve macrophyte richness and composition from 179 surface-sediment samples from arctic Siberian and alpine Chinese lakes and three representative lake cores. The surface-sediment dataset suggests that macrophyte richness and composition are mostly affected by temperature and conductivity, with highest richness when mean July temperatures are higher than 12°C and conductivity ranges between 40 and 400 μS cm−1. Compositional turnover during the Late Pleistocene/Holocene is minor in Siberian cores and characterized by a less rich, but stable emergent macrophyte community. Richness decreases during the Last Glacial Maximum and rises during wetter and warmer climate in the Late-glacial and Mid-Holocene. In contrast, we detect a pronounced change from emergent to submerged taxa at 14 ka in the Tibetan alpine core, which can be explained by increasing temperature and conductivity due to glacial runoff and evaporation. Our study provides evidence for the suitability of the trnL marker to recover modern and past macrophyte diversity and its applicability for the response of macrophyte diversity to lake-hydrochemical and climate variability predicting contrasting macrophyte changes in arctic and alpine lakes under intensified warming and human impact.