Gas and water samples were collected from coalbed methane (CBM) wells, rivers, and springs in the southern Junggar Basin (SJB). These samples were analyzed for gas composition, stable isotopes, 16s ribosomal ribonucleic acid sequence, chemical compositions, and radioisotopes. The objective of this study was to understand CBM genesis in the Junggar Basin, the reason for abnormal CO2 accumulation, the development of microbial communities, the source of coalbed water, and the timing of methanogenesis. The CBM genesis is complex in the SJB, but it is closely related to microbial activities. The stagnant zone, which experiences limited groundwater recharge, may represent a relatively closed system where CO2 is easily trapped and the residual CO2 becomes progressively enriched in 13C. Only two families of methanogens (i.e., Methanobacteriaceae and Methanospirillaceae) are present in the coalbed waters, indicating that CO2 reduction is the main pathway for generating microbial gas. The coalbed water samples from the Houxia and Manasi–Hutubi regions plot around the local meteoric water line (LMWL), indicating recharge by modern meteoric water and rivers. However, the samples from the Miquan and Fukang regions plot below the LMWL, reflecting older snowmelt water recharge. Isotopic dating indicates that the age of coalbed water in the Miquan and Fukang regions is 43.5–2000 ka. Early coalification and later hydrological events collectively determined the regional variations in CBM genesis and gas composition in the SJB.