Abstract Re–Os geochronology of oil may constrain the timing of oil formation and improve oil-source and oil–oil correlations. Typically, asphaltene (ASPH), the heaviest and most Re–Os rich oil fraction, from multiple oils within an oil field or a larger petroleum system are analyzed to obtain sufficient spread in Re–Os isotopic ratios, a mathematical necessity for precise Re–Os isochrons. Here we offer a new approach for Re–Os geochronology of oil based on isotopic analyses of different fractions within a single sample of crude oil. We studied three oils from the Gela oil field, southern Sicily, Italy, recovered from Triassic–Jurassic stratigraphic intervals (Streppenosa, Noto, and Sciacca Formations) within the Gela-1 well. ASPH (insoluble in n-alkane) and maltene (MALT, soluble in n-alkane) fractions of oil were separated using n-pentane, n-hexane, n-heptane and n-decane solvents. The ASPH contents of the Sciacca and Noto oils (26–33 wt%) are notably higher compared to the Streppenosa oil (7–12 wt% ASPH). We present an optimized Re–Os procedure with sample digestion in a high-pressure asher, followed by isotopic measurements using negative thermal ionization mass spectrometry. Very high metal contents of Gela oils allowed acquisition of precise Re–Os data. Systematic variations between the type of solvent used for ASPH precipitation and the ASPH content of the oil (also known from the literature) and the Re–Os contents of the ASPH and MALT fractions (first observed in this study) provide important practical applications for Re–Os analyses of oil. Most Re and Os (∼96–98%) in the Noto oil are hosted in the ASPH fraction. In contrast, a significant portion of Re and Os (∼33–34%) is stored in the MALT fraction of the lighter, but heavily biodegraded Streppenosa oil. Collectively, our new data on alkane distribution, hopane and sterane biomarkers, major and trace element contents, and Re–Os concentrations and isotopic ratios of the oils and their fractions support the presence of two oil families. Streppenosa oil, a heavily biodegraded oil generated at an early stage of thermal maturation from a shaly source rock, is clearly distinct from the Noto and Sciacca oils, both generated at peak maturation from a carbonate source rock with no subsequent biodegradation. Two Re–Os ages were obtained. Crude oil and ASPH from Noto and Sciacca oil yield a Model 1 isochron age of 27.5 ± 4.6 Ma with an initial 187 Os/ 188 Os of 3.89 ± 0.43 (MSWD = 1.6, n = 19). We interpret this Oligocene age as the time of initial oil generation, that was probably related to the onset of regional collisional tectonics in southern Sicily. ASPH and crude oil from the Streppenosa oil yield scattered data, perhaps related to effects of biodegradation. Streppenosa MALT yield a Model 1 age of 200.0 ± 5.2 Ma and initial 187 Os/ 188 Os of 1.39 ± 0.11 (MSWD = 0.52; n = 4). This age and initial Os-isotopic composition are consistent with rapid oil generation shortly after Late Triassic source-rock formation, perhaps driven by magmatic-related heating. Here we document a potentially powerful geochronological tool that can be applied to single samples of crude oil. Within each oil, the 187 Os/ 188 Os and most of the 187 Re/ 188 Os ratios in the MALT fractions are lower than in the corresponding ASPH fractions. Crude oils, being physical mixtures of ASPH and MALT, have intermediate Re–Os isotopic ratios. Hence, the needed spread in data points for a Re–Os isochron is obtained by analyzing components of a single crude oil. This approach eliminates pitfalls in interpretation of Re–Os data from multiple oils with possibly different generation, migration, or reservoir modification history.