Previous geochemical studies indicated that most natural gases dissolved in brines in Japan are of microbial origin, consisting of methane produced via carbonate reduction. However, some of those from gas fields in southwest Japan contain methane relatively enriched in 13C, whose origin remains to be clarified. To address this issue, chemical and isotopic analyses were performed on natural gases and brines from the gas fields in Miyazaki and Shizuoka prefectures, southwest Japan. Methane isotopic signatures (δ13C ≈ −68‰ to −34‰ VPDB; δ2H ≈ −183‰ to −149‰ VSMOW) suggest that these gases are of microbial (formed via carbonate reduction) or of mixed microbial and thermogenic origin. The relatively high δ2H-CH4 values and their relationship with the δ2H-H2O values argue against the possibility of their formation via acetate fermentation. The δ13C-CO2 values (≈−5‰), together with the slope of the correlation between δ2H-CH4 and δ13C-CH4 (Δδ2H-CH4/Δδ13C-CH4 ≈ 1), contradict the possibility of their formation via carbonate reduction followed by partial oxidation by methanotrophs. The 3He/4He ratios of the gases from Miyazaki (≈0.11–1.3 Ra) and their low correlation with δ13C-CH4 values do not support an abiogenic origin. It is inferred therefore that the high δ13C-CH4 values of natural gases dissolved in brines from gas fields in southwest Japan are indications of the contribution of thermogenic hydrocarbons, although whether abiogenic hydrocarbons contribute significantly to the gases from Shizuoka requires further investigation. This study has clarified that, for the future exploration of natural gases in southwest Japan, we should adopt the strategies for conventional thermogenic gas accumulations, such as checking the content, type and maturity of organic matter in the underlying sedimentary rocks.