Abstract: Abstract In the Manas River basin (MRB), groundwater salinization has become a major concern, impeding groundwater use considerably. Isotopic and hydrogeochemical characteristics of 73 groundwater and 11 surface water samples from the basin were analysed to determine the salinization process and potential sources of salinity. Groundwater salinity ranged from 0.2 to 11.91 g/L, and high salinities were generally located in the discharge area, arable land irrigated by groundwater, and depression cone area. The quantitative contributions of the evaporation effect were calculated, and the various groundwater contributions of transpiration, mineral dissolution, and agricultural irrigation were identified using hydrogeochemical diagrams and δD and δ18O compositions of the groundwater and surface water samples. The average evaporation contribution ratios to salinity were 5.87% and 32.7% in groundwater and surface water, respectively. From the piedmont plain to the desert plain, the average groundwater loss by evaporation increased from 7% to 29%. However, the increases in salinity by evaporation were small according to the deuterium excess signals. Mineral dissolution, transpiration, and agricultural irrigation activities were the major causes of groundwater salinization. Isotopic information revealed that river leakage quickly infiltrated into aquifers in the piedmont area with weak evaporation effects. The recharge water interacted with the sediments and dissolved minerals and subsequently increased the salinity along the flow path. In the irrigation land, shallow groundwater salinity and Cl− concentrations increased but not δ18O, suggesting that both the leaching of soil salts due to irrigation and transpiration effect dominated in controlling the hydrogeochemistry. Depleted δ18O and high Cl− concentrations in the middle and deep groundwater revealed the combined effects of mixing with paleo-water and mineral dissolution with a long residence time. These results could contribute to the management of groundwater sources and future utilization programs in the MRB and similar areas.