© 2017 American Chemical Society. The commonly held assumption that photodependent processes dominate H2O2production in natural waters has been recently questioned. Here, we present evidence for the unrecognized and light-independent generation of H2O2in groundwater of an alluvial aquifer adjacent to the Colorado River near Rifle, CO. In situ detection using a sensitive chemiluminescent method suggests H2O2concentrations ranging from lower than the detection limit (<1 nM) to 54 nM along the vertical profiles obtained at various locations across the aquifer. Our results also suggest dark formation of H2O2is more likely to occur in transitional redox environments where reduced elements (e.g., reduced metals and NOM) meet oxygen, such as oxic-anoxic interfaces. A simplified kinetic model involving interactions among iron, reduced NOM, and oxygen was able to reproduce roughly many, but not all, of the features in our detected H2O2profiles, and therefore there are other minor biological and/or chemical controls on H2O2steady-state concentrations in such aquifer. Because of its transient nature, the widespread presence of H2O2in groundwater suggests the existence of a balance between H2O2sources and sinks, which potentially involves a cascade of various biogeochemically important processes that could have significant impacts on metal/nutrient cycling in groundwater-dependent ecosystems, such as wetlands and springs. More importantly, our results demonstrate that reactive oxygen species are not only widespread in oceanic and atmospheric systems but also in the subsurface domain, possibly the least understood component of biogeochemical cycles.