Shale gas as an unconventional gas has made a significant impact on worldwide energy supply. The heterogeneity of the shale reservoir has a dramatic effect on the shale gas accumulation. The heterogeneity characterization of the Paleozoic Wufeng-Longmaxi Formation Shale in the Upper Yangtze Region is studied within a sequence stratigraphic framework by testing in situ gas content, organic matter, minerals, porosity, permeability, and the observation and description of core and thin sections, and data statistics. Results show that the shelf depocenter has higher porosity than the shelf margin and the permeability is obviously lower at the transitional zone compared to neighboring areas. The most obvious characteristic of organic matter and mineral composition is that the TOC and quartz content at the lower section of the formation deposited during transgressive systems tract is higher than that at the upper section of the formation deposited during highstand systems tract throughout the whole deposition zone. The laminae are primarily developed at the upper section of the formation. The natural fracture is mainly developed at the Qiyueshan fault zone and the Xuefeng Mountain fault zone. The primary controlling factors of shale gas accumulation are organic matter and porosity. The transgressive systems tracts with high organic matter content and porosity at the lower section of the formation are more favorable for gas accumulation. The shale gas resources and exploration potential are mainly concentrated in the shelf depocenter.