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Abstract 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.