The selenium (Se) content and the associated release mechanisms in both surface and groundwater have become a major concern worldwide over the past 30 years. Within the Hydrogeological Experimental Site of Poitiers (HESP), a large range of aqueous Se concentrations (from non-detectable to more than 30 ppb) is observed in a limited area (about 10ha), where water flows are highly characterized. This site thus consists of an interesting spot to better understand the release mechanisms of selenium into groundwater. The present study consists of an identification and a characterization of the lithological sources of Se within the HESP. Total rock analyses applied to core samples from different depths and wells demonstrated that selenium is concentrated in argillaceous sediments enriched with organic matter, pyrite and uranium that fulfill a part of the karst cavities developed within the Bajocian host rocks. Mineralogical and petrographic investigations highlighted the heterogeneity of these filling materials and showed the presence of successive deposits under different climatic conditions. Extensive characterizations dated the selected Se-rich samples from the Upper Cretaceous and converged to demonstrate the external and continental origin of the studied filling materials and their transformation after deposition under reduced conditions. Only indirect correlations allow considering an agreement between the Se history and the very mature organic matter identified in the argillaceous samples. This association will be further favored to determine the mechanisms releasing selenium into groundwater.