Fossil long-chain alkenones have been used for several decades to reconstruct past ocean surface water temperatures and gained recent interest as a paleotemperature proxy for continental lake settings. However, factors besides temperature can affect alkenone distributions in haptophyte algae, and alkenone compositions can differ between haptophyte species. Alkenone-biosynthesizing haptophyte algae are genetically much more diverse in lakes than in the marine realm, and species-level variations in alkenone compositions could have implications for alkenone paleothermometry. Here, we performed a paired analysis of alkenone distributions and haptophyte species compositions using ancient DNA in up to 270 ka-old sediments of Lake Van in Turkey to reveal a possible species-effect on fossil alkenone distributions and paleotemperature estimates. The same predominant haptophyte in Lake Van today prevailed also since the last ∼100 ka. However, a calibration of alkenone paleotemperature especially in the oldest analyzed intervals is complicated due to a more complex haptophyte species composition predominated by a haptophyte (LVHap_6), which is phylogenetically different from sequences recovered from currently existing lakes including Lake Van and from haptophyte species existing in culture. The predominance of LVHap_6 coincided with the presence of alkenone MeC38:3 and relatively high MeC37:3/4 (2.4) and MeC38:4/5 ratios (3.0). Uk37 index values in the sediment core over the last 270 ka reflect relative changes in past temperature and are additionally linked to haptophyte species composition. A sustained period of high salinity, as indicated by pore-water salinity measurements, could potentially have triggered the succession of haptophytes as sources of alkenones in Lake Van. •Alkenone analyses in a record from Eastern Anatolia covering the past 270 ka.•New insight into the influence of haptophyte species on alkenone distribution.•Semi-quantitative link between Uk37 index and past temperatures in Lake Van.