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Diatom-derived highly branched isoprenoid lipids (HBIs) are found extensively in marine sediments, but to date are only reported in a few lacustrine sediments. To expand on prior lake studies, we collected lake surface sediment samples, water samples, and filtered photic zone water from 50 lakes from the Great Plains to the northeastern United States. Samples were collected in May and June and a few sites were revisited in September and October. Studied lakes vary in climate, water chemistry (e.g., pH, salinity, alkalinity), size, and trophic states. They also vary in their diatom species compositions with 344 diatom taxa reported. We characterized HBI assemblages in each lake and found 11 different HBI compounds including one C20:0 HBI, five C20:1 HBI isomers, C21:0 HBI, C25:2 HBI, two C25:3 HBIs, and C25:4 HBI. C20:0 HBI was present in all but two lakes and was often the most abundant HBI present. HBIs were also detected in nearly all the water filter samples indicating they are produced in the photic zone. C20:0 HBI was present in all freshwater lakes, but not present or at very low con- centration in the highest salinity lakes, which were dominated by C21:0 HBI and C25 HBIs. Many of the lakes were dominated by diatom genera and species that are not known to be HBI-producing genera, suggesting there are unrecognized HBI-producing diatom taxa. This inventory, illustrating the widespread presence and diversity of HBIs from lakes across large differences in water chemistries and climate, further suggests that HBIs may be useful diatom biomarkers for paleoclimate applications.