Abstract. Methane is an important end product of degradation of organic matter in anoxic lake sediments. Methane is mainly produced by either reduction of CO₂ or cleavage of acetate involving different methanogenic archaea. The contribution of the different methanogenic paths and of the diverse bacteria and archaea involved in CH₄ production exhibits a large variability that is not well understood. Lakes in tropical areas, e.g. in Brazil, are wetlands with high potential impact on the global CH₄ budget. However, they have hardly been studied with respect to methanogenesis. Therefore, we used samples from 16 different lake sediments in the Pantanal and Amazon region of Brazil to measure production of CH₄, CO₂, analyze the content of ¹³C in the products and in intermediately formed acetate, determine the abundance of bacterial and archaeal microorgansisms and their community composition and diversity by targeting the genes of bacterial and archaeal ribosomal RNA and of methyl coenzyme M reductase, the key enzyme of methanogenic archaea. These experiments were done in the presence and absence of methyl fluoride, an inhibitor of acetoclastic methanogenesis. While production rates of CH₄ and CO₂ were correlated to the content of organic matter and the abundance of archaea in the sediment, values of ¹³C in acetate, CO₂, and CH₄ were related to the ¹³C content of organic matter and to the path of CH₄ production with its intrinsic carbon isotope fractionation. Isotope fractionation was small (average 10‰) for conversion of C_org to acetate-methyl, which was hardly further fractionated during CH₄ production. However, fractionation was strong for CO₂ conversion to CH₄ (average 75‰), which generally accounted for >50% of total CH₄ production. Canonical correspondence analysis did not reveal an effect of microbial community composition, despite the fact that it exhibited a pronounced variability among the different sediments.