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High levels of ammonium from anthropogenic sources threaten the quality of surface waters and groundwaters in some areas worldwide, but elevated ammonium levels of natural sources also have been identified. High levels of ammonium have been detected in both surface water and shallow groundwater of the Jianghan Plain, an alluvial plain of the Yangtze River. This study used N isotopes coupled with ancillary chemistry to identify ammonium in this region. Ammonium in the Tongshun River (up to 10.25 mg L) showed a sharp accumulation in the upstream and gradual attenuation in the downstream. The δN values of ammonium in the TSR were high and ranged narrowly from +12.5 to +15.4‰, suggesting an anthropogenic source that was septic effluent from industrial waste discharge. Sorption and nitrification were likely to respectively serve as the principal processes contributing to ammonium attenuation in different reaches of the downstream TSR. In shallow groundwater, high levels of ammonium (up to 14.10 mg L) occurred in a reducing environment. The narrow δN variation with low values (+2.3 to +4.5‰) in the lower aquifer suggested a natural source that was organic N mineralization. The δN values in the shallow aquitard exhibited a wide range from -1.8 to +9.4‰, owing to various sources. Two types of water in the shallow aquitard could be identified: (1) type-1 water with relatively longer residence time was similar to those in the aquifer where ammonium was mainly sourced from organic N mineralization; (2) type-2 water with shorter residence time was jointly affected by surface input, chemical attenuation and mineralization of organic N. The aquitard prevents prompt ammonium exchange between the surface and aquifer, and the shallower part of the aquitard provides a sufficient reaction time and an active reaction rate for ammonium removal.