- The concentrations and isotopic compositions of methane, higher alkanes, dissolved inorganic carbon (DIC) and CO 2 were studied in fresh groundwater, brines and springs along the Dead Sea Transform (DST), from the Hula Valley in the north to the Dead Sea (DS) basin in the south. Although the occurrence of methane along the DST was documented before, this is the first time that comprehensive research was conducted on the methane provenance and the post-genetic reactions involved. The methane stable carbon isotopic composition (δ 13 C CH4 ) shows a distinct geographic pattern. In the northern part of the DST studied, in the Hula valley and Lake Kinneret, where recent marshy and lacustrine environments exist, the methane source seems to be related to intense shallow depth methanogenesis. This microbial methane shows low 13 C values in the range of δ 13 C CH4 between − 58‰ and − 72‰ and high alkane ratios (C 1 /C 2 + C 3 ) between 100 and 1000. The isotope fractionation α CO2–CH4 of 1.065‰ suggests the domination of hydrogenotrophic methanogenesis rather than acetoclastic methanogenesis. In the waters of Hammat Gader and Tiberias hot spring, whose source is the Cretaceous aquifer, δ 13 C CH4 has high values of − 28‰ and − 56‰, respectively, with low alkane ratios of ~ 40 in both sites. The high δ 2 H CH4 values of Hammat Gader, in addition to the isotopic composition of the DIC and the difference between CO 2 and CH 4, reject the possibility of local microbially produced methane and suggest a sub-surface thermogenic source of an initial stage of thermal degradation of bituminous chalk of Senonian age. Similarly, methane in groundwater from the DS basin is also interpreted to be of thermogenic origin. In accordance with the local geology context in the DS basin of buried asphalt, and due to the low geothermal gradient in the region, it is speculated that methane is produced from slight heating of the asphalts. In general, it seems that there is a thermogenic methane contribution along the entire DST, which is locally masked by high microbial activity in shallow organic-rich environments, such as Lake Kinneret and the Hula basin.