This study summarizes the findings of our research on the genesis of methane, its content and distribution in permafrost horizons of different age and origin. Supported by reliable data from a broad geographical sweep, these findings confirm the presence of methane in permanently frozen fine-grained sediments. In contrast to the omnipresence of carbon dioxide in permafrost, methane-containing horizons (up to 40.0 mL kg−1) alternate with strata free of methane. Discrete methane-containing horizons representing over tens of thousands of years are indicative of the absence of methane diffusion through the frozen layers. Along with the isotopic composition of CH4 carbon (δ13C −64‰ to −99‰), this confirms its biological origin and points to in situ formation of this biogenic gas. Using 14C-labeled substrates, the possibility of methane formation within permafrost was experimentally shown, as confirmed by δ13C values. Extremely low values (near −99‰) indicate that the process of CH4 formation is accompanied by the substantial fractionation of carbon isotopes. For the first time, cultures of methane-forming archaea, Methanosarcina mazei strain JL01 VKM B-2370, Methanobacterium sp. strain M2 VKM B-2371 and Methanobacterium sp. strain MK4 VKM B-2440 from permafrost, were isolated and described. [ABSTRACT FROM AUTHOR]