The Tahe oil field is one of the largest deeply buried, multistage-charged Paleozoic oil fields in the Tarim Basin, northwestern China. Understanding the temporal evolution of Paleozoic oils is crucial for petroleum prospecting in this region. In this study, a combination of biomarker analysis, stable/radiogenic isotopic analysis, and Re-Os and U-Pb isotope geochronology is used to constrain the timing of oil accumulation in the Tahe oil field. Coexistence of unresolved complex mixture and intact n-alkane series in oil chromatograms suggests the presence of multistage oil accumulation processes in the studied area, and paleo-biodegradation occurred prior to the latest oil charge. Biomarker data show that the source rock for the latest accumulated oils was deposited in a marine reducing–weakly oxidizing environment. An early Permian oil generation event (276 ± 35 Ma) was suggested by Re-Os geochronology. This oil generation event is responsible for the late Permian–Late Triassic charge event (259.9–224.1 Ma), which is identified with in situ U-Pb isotopic analysis of calcites containing primary oil inclusions. These age data provide direct temporal constraints for the latest oil accumulation in the Tahe oil field. Furthermore, C-O-Sr-Os isotopic results show that hydrothermal fluid flowed during this oil accumulation event. Outcomes of this study unravel the Re-Os isotopic analysis of cogenetic crude oils and have the potential of direct dating the latest, hydrothermally linked oil accumulation in multistage-charged deep oil reservoirs.