With the targets of petroleum exploration transferred to the deep and ancient strata, abundant oil and gas resources have been found in the Lower Paleozoic and older strata in central and western China. Due to complex evolutionary processes including multiple episodes of hydrocarbon accumulation and ubiquitously accompanied by secondary alterations, significant uncertainties were remained for the generation time and accumulation processes of these revealed petroleum. In this paper, relative pure Re and Os elements existing in the asphaltene fractions of Lower Cambrian solid bitumen collected from the Guangyuan area, western Sichuan Basin, SW China and another Middle–Lower Ordovician heavy oils in the Aiding area of the Tahe oilfield in the Tarim Basin, NW China were successfully obtained by the sample pre-treatments, and Re–Os isotopic analysis was subsequently carried out for the dating of these two petroleum. The Re–Os isotopic composition indicates a generation time of Guangyuan bitumen to locate between 572 Ma and 559 Ma, corresponding to the late Sinian period of Neoproterozoic era. By the means of Re–Os isochron ages, initial 187Os/188Os ratios, and carbon isotopic compositions, the Lower Cambrian bitumen is supposed to be originated from source rocks of the Doushantuo Formation in the Sinian strata and subsequently migrated into the reservoirs of Dengying Formation. These previously reserved petroleum had been transformed to present bitumen by the destruction of reservoirs caused by tectonic uplift. The Re–Os dating results of Middle–Lower Ordovician heavy oil of Tarim Basin supposed that it was formed between 450 Ma to 436 Ma, corresponding to the Late Ordovician–Early Silurian system, and the generated petroleum is likely to migrate into the Middle–Lower Ordovician karst reservoirs to form early oil reservoirs. With tectonic uplift, these oil reservoirs were degraded and reformed to be present heavy oil reservoirs.