With increasing concerns of greenhouse gas (GHG) emissions from reservoirs, the debate surrounding whether hydropower qualifies as green energy has become increasingly contentious. However, quantifying the impact of dam construction on river GHG emissions still poses challenging due to the absence of direct observational data on river GHG emissions pre- and post-damming. In the present study, the differences in GHG diffusive fluxes from pre- and post-dammed river channels in the middle reaches of the Lancang River were quantified through three field campaigns in 2016, 2018, and 2023. The results indicate that reservoir construction increases the diffusive fluxes of GHG from the natural river, the total GHG emissions (expressed as carbon dioxide equivalent: CO2eq) from post-dammed river ranged from 234.42 to 527.55 mg CO2eq m−2 d−1 and were 1.4–5.2 times higher than that from pre-dammed river. However, the GHG diffusive fluxes from the reservoirs in the Lancang River remain below the average of global reservoirs. Moreover, river damming enhances the methane (CH4) emissions, the average value of CH4/CO2 ratio (in mg m−2 d−1) was 0.003 post-dam and was 13 times higher than that of pre-dam. Our results revealed that alterations in sediment distribution following reservoir construction emerge as a pivotal factor contributing to the variations in spatial distributions of GHG diffusive fluxes in the studied river channel. Overall, the findings of the study provide a primary and direct evidence of river damming impact on GHG emissions and underscore the necessity of long-term continuous in-situ measurements of GHG fluxes in the rivers undergoing hydropower development.