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• Temperature outcome on methanogenesis was investigated in austral lakes. • A 5 °C increase doubled methane production rates in freshwater sediments. • H 2 /CO 2 -based methanogenesis is more sensitive to temperature than acetate-based. • Microbial community structure exhibits high site-specific variability. • Methanosarcina and Methanobacterium respectively drove acetate- and H 2 /CO 2 -amendments. Freshwater ecosystems are responsible for an important part of the methane (CH 4) emissions which are likely to change with global warming. This study aims to evaluate temperature-induced (from 5 to 20 °C) changes on microbial community structure and methanogenic pathways in five sub-Antarctic lake sediments from Magallanes strait to Cape Horn, Chile. We combined in situ CH 4 flux measurements, CH 4 production rates (MPRs), gene abundance quantification and microbial community structure analysis (metabarcoding of the 16S rRNA gene). Under unamended conditions, a temperature increase of 5 °C doubled MPR while microbial community structure was not affected. Stimulation of methanogenesis by methanogenic precursors as acetate and H 2 /CO 2 , resulted in an increase of MPRs up to 127-fold and 19-fold, respectively, as well as an enrichment of mcrA -carriers strikingly stronger under acetate amendment. At low temperatures, H 2 /CO 2 -derived MPRs were considerably lower (down to 160-fold lower) than the acetate-derived MPRs, but the contribution of hydrogenotrophic methanogenesis increased with temperature. Temperature dependence of MPRs was significantly higher in incubations spiked with H 2 /CO 2 (c. 1.9 eV) compared to incubations spiked with acetate or unamended (c. 0.8 eV). Temperature was not found to shape the total microbial community structure, that rather exhibited a site-specific variability among the studied lakes. However, the methanogenic archaeal community structure was driven by amended methanogenic precursors with a dominance of Methanobacterium in H 2 /CO 2 -based incubations and Methanosarcina in acetate-based incubations. We also suggested the importance of acetogenic H 2 -production outcompeting hydrogenotrohic methanogenesis especially at low temperatures, further supported by homoacetogen proportion in the microcosm communities. The combination of in situ- , and laboratory-based measurements and molecular approaches indicates that the hydrogenotrophic pathway may become more important with increasing temperatures than the acetoclastic pathway. In a continuously warming environment driven by climate change, such issues are crucial and may receive more attention. [ABSTRACT FROM AUTHOR]