Type and origin of four methane gases from the Neogene fill of the Vienna Basin on the Slovak and Czech territory was estimated using chemical composition and hydrogen and carbon isotopic analysis. The gas from Malacky gas field (695 m deep) is composed almost exclusively of methane (C₂₊ = 0.02 vol. %) and is evidently of biogenic origin being rich in considerably light hydrogen and carbon isotopes (δD = -194 ‰; δ¹³C = -67 ‰). This gas is supposed to be formed in the Neogene not deeper than 1800 m. Isotopic composition of hydrogen (δD = -151 to –167 ‰) and organic carbon (δ¹³C = -39 to –43 ‰) of the gases from the Kuty gas field, Lab and Poddvorov oil- and gas fields (1368–1625 m) suggests their thermogenic (catagenetic) origin in pre-Neogene formations.