Wetland methane emissions in the permafrost regions of the Qinghai-Tibet Plateau is more sensitive to climate warming and can result in a positive climate feedback. Natural gas hydrate, as a potential methane source, may play a pivotal role in wetland methane emission in the permafrost regions. However, it was lacking of evidence. To determine the role of gas hydrate release in wetland methane emission, the two-year field monitoring of methane emitted from a hydrate drilling well, in near-surface soil free gas and low-level air was conducted at a typical gas hydrate reservior in the Qilian Mountains permafrost. The carbon isotope fractionation between CO2 and CH4 (εC) associated with carbon isotopic composition of methane (δ13CCH4) is used as a good tracer to identify methane sources of thermogenic origin or of microbial origin. The monitoring results of the gas hydrate drilling well DK-8 indicated a notable release of the deep gas hydrates occurred in April- May and resulted in the increase of methane content in low-level air. The significance of gas hydrate release in the permafrost region on local wetland methane emission as well as low-level air methane was confirmed by the seasonal variation of methane source of near-surface soil fluxes and low-level air. The thermogenically derived methane were identified as the dominant methane source in autumn and winter compared with increasing contribution of microbially derived methane in summer. The carbon isotopic signatures of tracing methane sources can provide more reliable evidence for gas hydrate release and its effect on the wetland methane emission in the Qilian Mountains permafrost.