Re–Os radiometric dating of crude oil can be used to constrain the timing of hydrocarbon generation, migration or charge. This approach has been successfully applied to marine petroleum systems; however, this study reports on its application to lacustrine-sourced natural crude oils. Oil samples from multiple wells producing from the Cretaceous Nantun Formation in the Wuerxun-Beier depression of the Hailar Basin in NE China were analysed. Subsets of the Re–Os data are compatible with a Cretaceous hydrocarbon generation event (131.1 ± 8.4 Ma) occurring within 10 Myr of deposition of the Nantun Formation source rocks. In addition, two younger age trends of 54 ± 12 Ma and 1.28 ± 0.69 Ma can be regressed from the Re–Os data, which may reflect the timing of subsequent hydrocarbon generation events. The Re–Os geochronometer, when combined with complementary age dating techniques, can provide direct temporal constraints on the evolution of petroleum system in a terrestrial basin.