The formation of groundwater from five aquifers (qp₂-³, qp₁ , n₂ ² , n₂ ¹ and n₁ ³ ) in the Nam Bo Plain was investigated by using isotope technique with the determination of water isotopic composition, namely δ² H and δ¹⁸O. For this purpose, 84 groundwater samples from the five aquifers (qp_2-3, qp₁ , n₂ ² , n₂ ¹ and n₁ ³ ) were taken by three transects AA: HoChiMinh city-CaMau, BB: Tay Ninh-HoChiMinh city and CC: Dong Thap-Tra Vinh. Additionally, 24 samples of precipitation in Ho Chi Minh city and 24 water samples from the Tien and Hau rivers were also taken during 2012-2014 for the study of their isotopic composition. The age of water from the aquifers was determined by the carbon-14 dating technique with δ13C correction based on the Gonfiantini’s model. Results of the isotopic composition of the samples showed that the groundwater in the region was originated from the local meteoric water as its isotopic composition was very close to that of the local meteorological water line. However, the age of the study water was determined predominantly to be old and ranged from 300 up to more than 40,000 years which suggests that the groundwater in the Nam Bo Plain is paleo-water. The Mann-Whitney statistical treatment with the mean δ¹⁸O value and its standard deviation for the two groups of groundwater in the western Nam Bo plain and water from both Tien and Hau rivers revealed that the river’s water did not have hydraulic interaction with water in the qp_2-3 aquifer. The same phenomena was found for most aquifers in the region, which means that the groundwater in the region has no or very weak recharge from surface. Since the groundwater resource in the Nam Bo Plain, especially in the western region is limited, it requires the local managers to pay particular attention to the groundwater exploitation in order to have enough clean water for the public as well as to maintain the water abstraction be sustainable. This is part of the statelevel scientific research project coded KC08.06/11-15