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Long chain alkenones (LCA) are temperature-sensitive lipids with great potential for quantitative reconstruction of past continental climate. We conducted the first survey for alkenone biomarkers from 55 different lakes in the Northern Great Plains and Nebraska Sand Hills of the United States. Among those surveyed, we found 13 lakes that contain LCAs in the surface sediments. The highest concentrations of alkenones in sediments are found in cold (mean annual air temperature ~11 °C versus 17 °C in our warmest sites), brackish to mesosaline (salinity = 8.5–9.7 g/L), and alkaline (pH = 8.4–9.0) lakes with high concentrations of sodium and sulfate. The dynamics of stratification and nutrient availability also appear to play a role in LCA abundance, as early spring mixing promotes a bloom of alkenone-producing haptophytes. Four of the alkenone-containing sites contain the C37:4 alkenone; however, we discovered an unprecedented lacustrine alkenone distribution in a cluster of lakes, with a total absence of C37:4 alkenone. We attribute this unusual composition to a different haptophyte species and show that the sulfate:carbonate ratio may control the occurrence of these two distinct populations. We created a new in-situ temperature calibration for lacustrine sites that contain C37:4 using a water-column calibration from Lake George, ND and show that UK37 is linearly correlated to lake water temperature (R2 = 0.74), but UK'37 is not. A number of lakes contain an unidentified compound series that elutes close to the LCAs, highlighting the importance of routine GC–MS examination prior to using lacustrine LCAs for paleotemperature reconstructions.