This study involved analyses of kerogen petrography, gas desorption, geochemistry, microporosity, and mesoporosity of the New Albany Shale (Devonian–Mississippian) in the eastern part of the Illinois Basin. Specifically, detailed core analysis from two locations, one in Owen County, Indiana, and one in Pike County, Indiana, has been conducted. The gas content in the locations studied was primarily dependent on total organic carbon content and the micropore volume of the shales. Gas origin was assessed using stable isotope geochemistry. Measured and modeled vitrinite reflectance values were compared. Depth of burial and formation water salinity dictated different dominant origins of the gas in place in the two locations studied in detail. The shallower Owen County location (415–433 m [1362–1421 ft] deep) contained significant additions of microbial methane, whereas the Pike County location (832–860 m [2730–2822 ft] deep) was characterized exclusively by thermogenic gas. Despite differences in the gas origin, the total gas in both locations was similar, reaching up to 2.1 cm³/g (66 scf/ton). Lower thermogenic gas content in the shallower location (lower maturity and higher loss of gas related to uplift and leakage via relaxed fractures) was compensated for by the additional generation of microbial methane, which was stimulated by an influx of glacial melt water, inducing brine dilution and microbial inoculation. The characteristics of the shale of the Maquoketa Group (Ordovician) in the Pike County location are briefly discussed to provide a comparison to the New Albany Shale.