As an important unconventional oil and gas resource, oil sands have been paid more and more attention all over the world. However, oil sands usually have some disadvantages such as strong heterogeneity and large differences in oil content, which leads to limited development efficiency. Based on the results of core, casting thin section and SEM observations, logging data, reservoir rock pyrolysis and soluble organic matter gas chromatography–mass spectrometry analysis, the mechanism of the oil content difference in the Cretaceous Upper McMurray Formation oil sands in the Mackay River area, Athabasca, Canada was studied. The diagenesis of the Mackay River oil sands is weak and there is no cement. The rock characteristics are mainly determined by three parameters: grain size, sorting and matrix content. The rock types are divided into extremely fine sandstone, fine sandstone, greywacke and siltstone. According to logging and core water saturation data, the oil sands reservoir can be evaluated as the gas, water, oil and poor oil interval, which are mainly composed of extremely fine sandstone, fine sandstone, greywacke and siltstone, respectively. The biomarker characteristics show that bitumen derives from the same source kitchen and there is no difference in maturity. Therefore, the main reason for the difference in oil content may be the difference in degradation degree and the amount of oil charged due to the diversity in reservoir petrological. Fine sandstone has good connectivity and is easy to be affected by degradation such as washing and oxidation, and usually forms the water interval. The siltstone is dense, and the early oil charging has difficulty reaching completion and this usually forms the poor interval. The properties of greywacke lie somewhere in between, the degree of degradation is relatively low and without affecting the early charging of oil, so it usually forms the oil interval.