The Mohe permafrost in northeast China possesses favorable subsurface ambient temperature, salinity, Eh values and pH levels of groundwater for the formation of microbial gas, and the Mohe Basin contains rich organic matter in the Middle Jurassic dark mudstones. This work conducted gas chromatography and isotope mass spectrometry analyses of nearly 90 core gas samples from the Mk-2 well in the Mohe Basin. The results show that the dryness coefficient (C1/C1–5) of core hydrocarbon gas from approximately 900 m intervals below the surface is larger than 98%, over 70% of the δ13C values of methane are smaller than ?55‰, and almost all δD values of methane are smaller than ?250‰, indicative of a microbial origin of the gas from almost 900 m of the upper intervals in the Mohe permafrost. Moreover, the biomarker analyses of 72 mudstone samples from the Mohe area indicate that all of them contain 25-norhopane series compounds, thereby suggesting widely distributed microbial activities in the permafrost. This work has confirmed the prevailing existence of microbial gas in the Mohe area, which may be a potential gas source of gas hydrate formation in the Mohe permafrost. This result is of great significance to gas hydrate accumulation in the permafrost across China.