Alkenones are C35–C42 polyunsaturated ketone lipids that are commonly employed to reconstruct changes in sea surface temperature. However, their use in coastal seas and saline lakes can be hindered by species-mixing effects. We recently hypothesized that freshwater lakes are immune to species-mixing effects because they appear to exclusively host Group I haptophyte algae, which produce a distinct distribution of alkenones with a relatively consistent response of alkenone unsaturation to temperature. To evaluate this hypothesis and explore the geographic extent of Group I haptophytes, we analyzed alkenones in sediment and suspended particulate matter samples from lakes distributed throughout the mid- and high latitudes of the Northern Hemisphere (n=30). Our results indicate that Group I-type alkenone distributions are widespread in freshwater lakes from a range of different climates (mean annual air temperature range: −17.3–10.9 °C; mean annual precipitation range: 125–1657 mm yr−1; latitude range: 40–81°N), and are commonly found in neutral to basic lakes (pH > 7.0), including volcanic lakes and lakes with mafic bedrock. We show that these freshwater lakes do not feature alkenone distributions characteristic of Group II lacustrine haptophytes, providing support for the hypothesis that freshwater lakes are immune to species-mixing effects. In lakes that underwent temporal shifts in salinity, we observed mixed Group I/II alkenone distributions and the alkenone contributions from each group could be quantified with the RIK37 index. Additionally, we observed significant correlations of alkenone unsaturation (U37 K) with seasonal and mean annual air temperature with this expanded freshwater lakes dataset, with the strongest correlation occurring during the spring transitional season (U37 K=0.029⁎T−0.49; r2=0.60; p<0.0001). We present new sediment trap data from two lakes in northern Alaska (Toolik Lake, 68.632°N, 149.602°W; Lake E5, 68.643°N, 149.458°W) that demonstrate the highest sedimentary fluxes of alkenones in the spring transitional season, concurrent with the period of lake ice melt and isothermal mixing. Together, these data provide a framework for evaluating lacustrine alkenone distributions and utilizing alkenone unsaturation as a lake temperature proxy.