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The origin and chemistry of the groundwater in the middle Pleistocene (qp23), lower Pleistocene (qp1), upper Pliocene (n22), lower Pliocene (n21), and the Miocene (n13) in the Mekong river delta (MKRD) were investigated by using isotopic and geochemical techniques. The origin of the groundwater was evaluated based on the composition of the water stable isotopes (delta2H and delta18O) in the local precipitation, in water from the rivers system, and in the groundwater samples. The hydraulic interaction between the surface water and the groundwater as well as between the aquifers was assessed by a statistical treatment for the mean and standard deviation of the delta18O signature and based on the 14C-ages of the water samples taken from different aquifers. The salinization of groundwater in the deep aquifers was investigated using the delta18O signature combined with the geochemical composition of the water samples. It was revealed that the groundwater in the deep aquifers in the MKRD could be divided into two groups. The first group is fresh and represents the regional precipitation with the long travelling time ranging from older than 100 years to older than 40 ka BP (kilo years Before Present). The second group is the regional precipitation that is recharged from the remote areas mixed with the seawater. Statistical treatment with the mean delta18O using the Mann-Whitney test showed that the water from the Mekong river system did not or very weakly recharged the deep aquifers. The groundwater in the deep aquifers in that region was likely to be connate, so that fresh groundwater resource in the region seemed to be limited. The chemistry of the groundwater in the study region is controlled by the incongruent dissolution of the Mg-calcite as well as sulfate and iron oxy-hydroxide reductions by organic matters presented in the aquifer sediment. The groundwater in the deep aquifers in the MKRD from some locations was saline, but the salinity in most aquifers was thought to result from the migration of saline water entrapped in the marine sediment pores to the fresh water in the aquifers. Meanwhile, in other locations, the salinity was suggested to result from the salt intrusion due to the over-abstraction rate, as it was evident from the delta18O vs. [Cl-] relationship, or due to the up conning of saline water from the deeper aquifers to the upper ones. Particular measures must be developed for the better management of groundwater in the MKRD to ensure a sustainable resource of freshwater being supplied to the local population in future.