An understanding of the temporal evolution of a petroleum system is fundamental to interpreting where hydrocarbons may be trapped in the subsurface. However, traditional exploration methods provide few absolute constraints on the timing of petroleum generation. Here we show that 187Re/187Os geochronology may be applied to natural crude oil seepage to determine when petroleum generation occurred in offshore sedimentary basins. Using asphaltites collected from the South Australian coastline, our determined Re-Os age (68 ± 15 million years ago) is consistent with their derivation from a Late Cretaceous source rock in the nearby Bight Basin, an interpretation similarly favoured by source-specific biomarker constraints. Furthermore, the calculated initial 187Os/188Os composition of the asphaltites, a value inherited from the source rock at the time of oil generation, suggests that the source rock represents the later stage of Oceanic Anoxic Event 2. Our results demonstrate a new approach to identifying the origin of crude oils encountered in coastal environments by providing direct constraints on the timing of petroleum generation and potential source rock intervals in poorly characterised offshore sedimentary basins prior to exploratory drilling.