The early Eocene Okanagan Highland fossil sites of Washington (USA) and British Columbia (Canada) contain exquisitely preserved plant and insect fossils that showcase a critical time and place in the evolution of the Northern Hemisphere temperate deciduous biome. A comprehensive understanding of the dynamics of fossil deposition and preservation at these sites is not fully resolved but is critical for reliable reconstructions of these ancient forests. To expand on previous interpretations (e.g., deep, stratified, anoxic lake bottoms) and address uncertainties about the environment of deposition (e.g., distance to shore, influence of diatoms), we analyzed sediment samples from three Okanagan Highland fossil sites—McAbee, Falkland, and Driftwood Canyon—for organic biomarkers, their stable carbon isotopic compositions, and glycerol dialkyl glycerol tetraethers (GDGTs; at McAbee only). Terpenoids suggest relative trends in gymnosperm abundance between sites that agree with prior macrofossil evidence, though absolute values may overestimate local gymnosperm abundance. A combination of biomarker evidence indicates a predominantly autochthonous aquatic source (e.g., diatoms) for organic matter in shale and mudstone samples, even contributing to long chain n-alkanes and likely to branched GDGTs, which are often assumed to be terrestrially sourced. In combination with biomarker evidence for anoxia and stratification, fossiliferous shales are interpreted to have been deposited offshore in deep and mesotrophic lakes that were thermally stratified with an anoxic hypolimnion, away from in-flowing tributaries, while a coal horizon at Driftwood Canyon was deposited in a shallower, eutrophic, anoxic wetland. Anoxic conditions likely minimized some degradation-based biases and promoted high quality fossil preservation. Deposition of sediment and fossil remains offshore and away from inflowing tributaries suggest fossil plants were locally sourced but highlights the need for careful consideration of transport-induced biases.