Hydrocarbon souring represents a significant safety and corrosion challenge to the oil and gas industry. H2S may originate from geochemical or biogenic sources, although its source is rarely discerned. Biocides are sometimes utilized during well operations to prevent or inhibit H2S generation. Here we develop a regional temperature map showing that downhole temperatures in Bakken reservoir wells equal or exceed the upper known temperature limit for microbial life. Attempts to extract microbial DNA from produced water yielded little to no detectable quantities. Stable isotope analysis yielded 34Sδ values from 4.4 to 9.8‰, suggesting souring had a geochemical origin. Under Bakken reservoir conditions, anhydrite can react with hydrocarbons to form H2S. Anhydrite present near the sour areas studied could be the underlying geochemical source creating this H2S. In cases of geochemical souring, reevaluation of the need for biocide addition may provide significant reductions in both operational costs and overall environmental footprint.