Abstract Understanding methane (CH 4 ) 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 CH 4 emissions from 120 thermokarst lakes in 30 clusters along a 1100 km transect on the Tibetan Plateau. We find that thermokarst lakes have high CH 4 emissions during the ice-free period (13.4 ± 1.5 mmol m −2 d −1 ; mean ± standard error) across this alpine permafrost region. Ebullition constitutes 84% of CH 4 emissions, which are fueled primarily by young carbon decomposition through the hydrogenotrophic pathway. The relative abundances of methanogenic genes correspond to the observed CH 4 fluxes. Overall, multiple parameters obtained in this study provide benchmarks for better predicting the strength of permafrost carbon-climate feedback in high-altitude permafrost regions.