The Kuqa Depression is rich in oil and gas resources and serves as a key production area in the Tarim Basin. However, controversy persists over the genesis of oil and gas in the various structural zones of the Kuqa Depression. This study employs natural gas composition analysis, gas carbon isotope analysis and gold pipe thermal simulation experiments, to comprehensively analyze the differences in the genesis and sources of hydrocarbon gas fluid from the eastern and western Kuqa Depression. The results show that the Kuqa Depression is dominated by alkane gas, with an average gas drying coefficient of 95.6, with nitrogen and carbon dioxide as the primary non-hydrocarbon gases. The average of δ13C1, δ13C2 and δ13C3 values in natural gas are −27.70‰, −20.43‰ and −21.75‰, respectively. Based on comprehensive natural gas geochemical maps, the CO2 in the natural gas from the Tudong and Dabei areas, as well as the KT-1 well of the Kuqa Depression, is thought to be of organic origin. Additionally, natural gas formation in the Tudong area is relatively simple, consisting entirely of thermally generated coal gas derived from the initial cracking of kerogen. The natural gas in the KT-1 well and the Dabei area are mixed gasses, formed by the initial cracking of kerogen from highly evolved lacustrine and coal-bearing source rocks, exhibiting characteristics resembling those of crude oil cracking gas. The methane (CH4) content of natural gas in the Dabei area is high and the carbon isotopes are unusually heavy. Considering the regional geological background, potential source rock characteristics and geochemical features may be related to the large-scale invasion of dry gas contributed by CH4 from highly evolved, underlying coal-bearing source rocks. Consequently, the CH4 content in the mixed gas is generally high (Ln (C1/C2) can reach up to 5.38), while the relative content of heavy components is low, though remains relatively unchanged. Thus, the map of the relative content of heavy components still reflects the characteristics of the original gas genesis (initial cracking of kerogen). Mixed-source gas was analyzed using thermal simulation experiments and natural gas composition ratio diagrams. The contributions of natural gas from deep, highly evolved coal-bearing source rocks in the KT-1 well and the Dabei area accounted for more than 90% and approximately 60%, respectively. This analysis provides theoretical guidance for natural gas exploration in the research area.