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Groundwater plays a vital role in the arid Southern Gobi Region (SGR) of Mongolia. It is the only source of water supply and is largely utilized for mining operations, such as those at Oyu Tolgoi Mine. The area is expected to face severe water shortages for mining and drinking water, which may be related to changes in groundwater recharge due to climate change. Understanding the geochemical evolution of groundwater and recharge mechanisms is thus paramount for water resource management. In this study, for the first time, chemical and isotopic approaches have been used to characterize the groundwater origin and its associated recharge processes in the Gunii Khooloi basin, which is located in the SGR. Shallow groundwater is mainly characterized by Na(HCO3) and NaCl type; however, Gunii Khooloi aquifer is classified as NaCl type with high electrical conductivity. The stable isotope composition of the water in the deep Cretaceous confined aquifer shows depletion in H-2 and O-18 relative to modern rainfall and the Quaternary shallow aquifer, which indicates a weak interaction between the two aquifers. Modern groundwater was identified in the shallow streambed aquifer, probably due to the direct infiltration of rainfall. On the other hand, C-14 ages in the deep groundwater range from 2,800 to 33,500 years, which suggests that this is paleowater that was recharged during the glacial age. The results indicate that there is a need for strategic groundwater management in the Gunii Khooloi basin.