Much of the world’s heavy oil is found in Cretaceous reservoir rocks due to a combination of tectonic, climatic, geological, and biological factors. Here we study Cretaceous oil sands from the Neuquén Basin (Argentina), Sergipe-Alagoas Basin (Brazil), Alberta (Canada), Dahomey Basin (Nigeria), Uinta Basin (USA), Western Moray Firth Basin (United Kingdom), and Wessex Basin (United Kingdom) to improve our understanding of the origin of the heavy oils. Our results indicate that the oils were generated as conventional light oil, which later degraded into heavy oils, rather than thermally cracked oils from over matured source rocks. All the studied Cretaceous oil sands are enriched in the polar fraction, and the total ion current (TIC) fragmentogram of the saturate fractions show unresolved complex mixture (UCM) humps indicating that the oils have undergone biodegradation. Sterane data for the Cretaceous oil sands show a selective increase in the C29 regular steranes relative to C27 and C28 regular sterane, which is also consistent with biodegradation. There is also evidence for diasterane degradation in some samples which are related, suggesting severe biodegradation. The trisnorhopane thermal maturity indicator showed that the Cretaceous oil sands have thermal maturity levels equivalent to 0.66–1.32% Ro, consistent with an early to late oil window. 25-norhopanes were not detected in any of the studied Cretaceous oil sands despite sterane degradation. This strongly suggests that biodegradation in the Cretaceous oil sands occurred at shallow depths rather than at greater depths. Pyrite associated with the Cretaceous oil sands was found to be consistently isotopically light. The isotopic fractionation between these pyrites and contemporary seawater sulfate was calculated using the mean δ34S values and the established seawater composition curve. This fractionation exceeded the maximum known kinetic isotope fractionation of ~20‰ that is possible from non-biogenic mechanisms, such as thermochemical sulfate reduction. This strongly suggests that the pyrite precipitated from an open system by means of microbial sulfate reduction as part of the biodegradation process.