Understanding methane (CH4) emission from thermokarst lakes is crucial for predicting the impacts of abrupt thaw on the permafrost carbon-climate feedback. However, observational evidence, especially from high-altitude permafrost regions, is still scarce. Here, by combining field surveys, radio- and stable-carbon isotopic analyses, and metagenomic sequencing, we present multiple characteristics of CH4emissions from 120 thermokarst lakes in 30 clusters along a 1100 km transect on the Tibetan Plateau. We find that thermokarst lakes have high CH4emissions during the ice-free period (13.4 ± 1.5 mmol m−2d−1; mean ± standard error) across this alpine permafrost region. Ebullition constitutes 84% of CH4emissions, which are fueled primarily by young carbon decomposition through the hydrogenotrophic pathway. The relative abundances of methanogenic genes correspond to the observed CH4fluxes. Overall, multiple parameters obtained in this study provide benchmarks for better predicting the strength of permafrost carbon-climate feedback in high-altitude permafrost regions.