Direct dating of petroleum systems by hydrocarbon or associated authigenic minerals is crucial for petroleum system analysis and hydrocarbon exploration. The precipitation of authigenic pyrite in petroliferous basins is commonly genetically associated with hydrocarbon generation, migration, accumulation, or destruction. Pyrite rubidium-strontium (Rb-Sr) isotope dilution thermal ionization mass spectrometry (ID-TIMS) is a well-developed technique, and its successful application for high-temperature ore systems suggests that this dating method has the potential to directly date key processes in the low-temperature petroleum systems. Rb-Sr data for pyrites in two Ordovician carbonate rock specimens collected from ~4952 m in the YD-2 well in the Yudong region, northern Tarim Basin (NW China), yield two identical isochron ages within analytical uncertainties: 206 ± 13 (2σ) and 224 ± 28 Ma (2σ). SEM investigations demonstrate that Rb and Sr atoms mainly reside in the crystal lattice of the pyrites due to the absence of fluid and mineral inclusions. The rigorous Rb-Sr isochron relations documented in the samples indicate that such residency can result in sufficient Rb/Sr fractionation and initial Sr isotopic homogenization for geochronology. In addition, the closure temperature (227–320 °C) for the Rb-Sr isotope system in pyrite is higher than the precipitation temperature for pyrite in petroleum-related environments (below 200 °C), suggesting that the Rb-Sr age of pyrite was not overprinted by post-precipitation alteration. Integrating the lead-strontium-sulfur isotopes of the pyrites with burial history analysis, the ages are interpreted as the timing of alteration of the paleo-oil pool by a hydrothermally-triggered thermochemical sulfate reduction process. This study, for the first time, demonstrates that Rb-Sr pyrite geochronology, combined with radiogenic and stable isotopic analyses, can be a useful tool to evaluate the temporal evolution of oil pools. This approach bears great potential for dating of petroleum systems anywhere else in the world.