Lacustrine sedimentary successions provide exceptionally high-resolution records of continental geological processes, responding to tectonic, climatic and magmatic influences. These successions are therefore essential for correlating geological and climatic phenomena across continents and furthermore the globe. Producing accurate geochronological frameworks within lacustrine strata is challenging because the stratigraphy is often bereft of biostratigraphy and directly dateable tuff horizons. The rhenium–osmium (Re–Os) geochronometer is a well-established tool for determining precise and accurate depositional ages of marine organic-rich rocks. Lake systems with stratified water columns are predisposed to the preservation of organic-rich rocks and thus should permit direct Re–Os geochronology of lacustrine strata. We present Re–Os systematics from one of the world's best documented lacustrine systems, the Eocene Green River Formation, providing accurate Re–Os depositional dates that are supported by Ar–Ar and U–Pb ages of intercalated tuff horizons. Precision of the Green River Formation Re–Os dates is controlled by the variation in initial 187Os/188Os and the range of 187Re/188Os ratios, as also documented in marine systems. Controls on uptake and fractionation of Re and Os are considered to relate mainly to depositional setting and the type of organic matter deposited, with the need to further understand the chelating precursors of Re and Os in organic matter highlighted. In addition to geochronology, the Re–Os data records the 187Os/188Os composition of lake water (1.41–1.54) at the time of deposition, giving an insight into continental runoff derived from weathering of the geological hinterland of the Green River Formation. Such insights enable us to evaluate fluctuations in continental climatic, tectonic and magmatic processes and provide the ability for chemostratigraphic correlation combined with direct depositional dates. Furthermore, initial 187Os/188Os values can be used as a diagnostic tool to distinguish between lacustrine and marine depositional settings when compared to known oceanic 187Os/188Os values.