The western Hubei–eastern Chongqing area is an important prospective zone for oil and gas exploration in the central Yangtze area. Three representative structures, the Xinchang structure, Longjuba gas-bearing structure and the Jiannan gas field, were selected to analyze biomarker parameters in marine strata and to examine various types of natural gas and hydrocarbon sources. Fluid inclusions; carbon, oxygen, and strontium isotopic characteristics; organic geochemical analysis and simulation of hydrocarbon generation and expulsion history of source rocks were used for tracing fluid migration paths in marine strata of the study area. The Carboniferous-Triassic reservoirs in three typical structures all experienced at least two stages of fluid accumulation. All marine strata above the early Permian were shown to have fluids originating in the Permian rocks, which differed from the late stage fluids. The fluids accumulated in the late Permian reservoirs of the Xinchang structure were Cambrian fluids, while those in the late Carboniferous reservoirs were sourced from a combination of Silurian and Cambrian fluids. A long-distance and large-scale cross-formational flow of fluids destroyed the preservation conditions of earlier accumulated hydrocarbons. A short-distance cross-formational accumulation of Silurian fluids was shown in the late Permian reservoirs of the Longjuba structure with favorable hydrocarbon preservation conditions. The fluid accumulation in the Carboniferous reservoirs of the Jiannan structure mainly originated from neighboring Silurian strata with a small amount from the Cambrian strata. As a result, the Jiannan structure was determined to have the best preservation conditions of the three. Comparative analysis of fluid migration paths in the three structures revealed that the zone with a weaker late tectonism and no superimposition and modification of the Upper and Lower Paleozoic fluids or the Upper Paleozoic zone with the fluid charging from the Lower Paleozoic in the western Hubei–eastern Chongqing area are important target areas for future exploration.