Groundwater forms the main freshwater supply in arid and semi-arid areas, and contamination of this precious resource is complicated by the slow rate of recharge in these areas. Nitrate contamination of groundwater is a global water quality problem, as it entails threat to human health as well as aquatic ecosystems. Source identification of contamination is the cornerstone and a prerequisite for any effective management program of water quality. Stable isotope composition of the dissolved nitrate (delta N-15-NO3- and delta O-18-NO3-) has been applied to identify NO3- sources and the main transformation processes in the upper aquifer system (A1/2, A4, and B2/A7 aquifers) in the Wadi Shueib catchment area, Jordan. Moreover, the stable isotope compositions of the groundwater (delta H-2-H2O and delta O-18-H2O) in conjunction with the groundwater hydrochemistry were integrated to investigate the origin and evolution of the groundwater. Results revealed that groundwater in the study area is fresh and hard-very hard water, and mainly a Ca-Mg-Cl type. NO3- concentration was in the range of 7.0-74.0 mg/L with an average of 37.0 mg/L. Most of the samples showed concentration higher than the natural background concentration of NO3- (5.0-10.0 mg/L). The delta H-2-H2O and delta O-18-H2O values indicated that the groundwater is meteoric, and of Mediterranean origin, with a strong evaporation effect. The delta N-15-NO3- values ranged between 6.0% and 11.3% with an average of 8.7%, and the delta O-18-NO3- values ranged between 1.6% and 5.9% with an average of 3.4%. These values are in conformity with the stable isotope composition of nitrate derived the nitrification of wastewater/manure, and soil NH4. Nitrification and denitrification are the main transformation processes affecting nitrogen species. Statistical analysis revealed no significant differences in the delta H-2-H2O and delta O-18-H2O values, and delta N-15-NO3- and delta O-18-NO3- values for the three aquifers (A1/2, A4, and B2/A7), indicating that the groundwater of these aquifers has the same origin, and a common source of pollution.