This study uses stable noble gases' (He, Ne, Ar, Kr, Xe) volume fractions and isotopic ratios from Antrim Shale natural gas to assess compositional variability and vertical fluid migration within this reservoir, in addition to distinguishing between the presence of thermogenic versus biogenic methane. R/Ra values, where R is the measured 3He/4He ratio and Ra is the atmospheric value of 1.384 ± 0.013 × 10− 6, vary from 0.01 to 0.34 suggesting a largely dominant crustal 4He component with minor atmospheric and mantle contributions. Crustal 21Ne, 40Ar and 136Xe contributions are also present but the atmospheric component is largely dominant for these gases. Crustal contributions for 21Ne, 40Ar and 136Xe vary between 1.1% and 12.5%, between 0.7% and 19% and between 0.1% and 2.7%, respectively. A few samples present higher than atmospheric 20Ne/22Ne ratios pointing to the presence of a small mantle Ne component. High horizontal and vertical variability of noble gas signatures in the Antrim Shale are observed. These are mainly due to variable noble gas input from deep brines and, to a smaller extent, variable in-situ production within different layers of the Antrim Shale, in particular, the Lachine and Norwood members. Estimated 4He ages, considering external 4He input for Antrim Shale water, vary between 0.9 ka and 238.2 ka and match well for most samples with the timing of the major Wisconsin glaciation, suggesting that Antrim Shale water was influenced by glaciation-induced recharge. Consistency between measured and predicted 40Ar/36Ar ratios assuming Ar release temperatures ≥ 250 °C supports a thermogenic origin for most of the methane in these samples. This thermogenic methane is likely to originate at greater depths, either from the deeper portion of the Antrim Shale in the central portion of the Michigan Basin or from deeper formations given that the thermal maturity of the Antrim Shale in the study area is rather low.