Coalbed gases and waters from exploratory and production gas wells in the southeastern Illinois Basin were sampled to assess geochemically the origin of coalbed gases, with an emphasis on the Springfield and Seelyville Coal Members that are commercially targeted for coalbed methane production in Indiana. On-line analyses of hydrocarbon gases methane to butanes (C1, C2, C3, n-C4, i-C4) and CO2 yielded gas concentrations, plus ??D and ??13C values. The low thermal maturity of Indiana coals with vitrinite reflectance R0 ??? 0.6% is in agreement with an overwhelmingly biogenic isotopic signature of coalbed gases containing ???96% methane generated via bacterial CO2-reduction. In contrast, thermogenic gas was generated in the stratigraphically equivalent coal beds in western Kentucky's Rough Creek Graben zone where higher maturities of up to R0 ??? 0.8% were reached owing to tectonic and hydrothermal activity. No secondary biogenic methane was observed in more mature western Kentucky coal beds where greater burial depth limits the recharge of meteoric water. Biogenic and thermogenic coalbed gases represent two end-members that are compositionally and isotopically distinct. Microbial biodegradation of thermogenic C2+ hydrocarbon gases in Indiana coal beds preferentially targets C3 and introduces isotope fractionation whereby remaining C3 is enriched in deuterium and 13C.