Long chain alkenones (LCAs) are potential biomarkers for quantitative paleotemperature reconstructions from lacustrine environments. However, progress in this area has been severely hindered by the lack of culture studies of haptophytes responsible for alkenone distributions in lake sediments: the predominance of C37:4 LCA. Here we report the first enrichment culturing of a novel haptophyte phylotype (Hap-A) from Lake George, ND that produces predominantly C37:4-LCA. Hap-A was enriched from its resting phase collected from deep sediments rather than from water column samples. In contrast, enrichments from near surface water yielded a different haptophyte phylotype (Hap-B), closely related to Chrysotila lamellosa and Pseudoisochrysis paradoxa, which does not display C37:4-LCA predominance (similar enrichments have been reported previously). The LCA profile in sediments resembles that of Hap-A enrichments, suggesting that Hap-A is the dominant alkenone producer of the sedimentary LCAs. In enrichments, excess lighting appeared to be crucial for triggering blooms of Hap-A. Both and indices show a linear relationship with temperature for Hap-A in enrichments, but the relationship appears to be dependent on the growth stage. Based on 18S rRNA gene analyses, several lakes from the Northern Great Plains, as well as Pyramid Lake, NV and Tso Ur, Tibetan Plateau, China contain the same two haptophyte phylotypes. The Great Plains lakes show the Hap-A-type LCA distribution, whereas Pyramid and Tso Ur show the Hap-B type distribution. Waters of the Great Plain lakes are dominated by sulfate, whereas those Pyramid and Tso Ur are dominated by carbonate, suggesting that the sulfate to carbonate ratio may be a determining factor for the competitiveness of the Hap-A and Hap-B phylotypes in natural settings.