During the Messinian, 6 m.y. ago, massive sea-level fall and widespread deposition of evaporites occurred in the Mediterranean Sea when it became isolated from the world oceans. Here we present the first hydrogen isotope data from individual sedimentary biomarkers, n-alkanes and isoprenoids, that tracked climatically driven hydrographic changes in response to extreme evaporation during the Messinian salinity crisis. The stable hydrogen and carbon isotope compositions of these biomarkers show a range of 160‰ in δD values and 14‰ in δ13C values, and roughly covary. This indicates that the source waters of the biomarkers were therefore in some cases extremely enriched in deuterium, having average δD as great as +66‰ VSMOW (Vienna standard mean ocean water). Such values are only known from desert climates today. Because the offset between the δD values of n-alkanes and isoprenoids preserved in the Miocene sedimentary rocks is similar to the offset found in modern biological samples, we conclude that diagenesis did not significantly affect the primary deuterium concentrations.