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Existing delta H-2 and delta O-18 values for precipitation and surface water in the Nile Basin were used to analyze precipitation inputs and the influence of evaporation on the isotopic signal of the Nile River and its tributaries. The goal of the data analysis was to better understand basin processes that influence seasonal streamflow for the source waters of the Nile River, because climate and hydrologic models have continued to produce high uncertainty in the prediction of precipitation and streamflow in the Nile Basin. An evaluation of differences in precipitation delta H-2 and delta O-18 values through linear regression and distribution analysis indicate variation by region and season in the isotopic signal of precipitation across the Nile Basin. The White Nile Basin receives precipitation with a more depleted isotopic signal compared to the Blue Nile Basin. The hot temperatures of the Sahelian spring produce a greater evaporation signal in the precipitation isotope distribution compared to precipitation in the Sahara/Mediterranean region, which can be influenced by storms moving in from the Mediterranean Sea. The larger evaporative effect is reversed for the two regions in summer because of the cooling of the Sahel from inflow of Indian Ocean monsoon moisture that predominantly influences the climate of the Blue Nile Basin. The regional precipitation isotopic signals convey to each region’s streamflow, which is further modified by additional evaporation according to the local climate. Isotope ratios for White Nile streamflow are significantly altered by evaporation in the Sudd, but this isotopic signal is minimized for streamflow in the Nile River during the winter, spring and summer seasons because of the flow dominance of the Blue Nile. During fall, the contribution from the White Nile may exceed that of the Blue Nile, and the heavier isotopic signal of the White Nile becomes apparent. The variation in climatic conditions of the Nile River Basin provides a means of identifying mechanistic processes through changes in isotope ratios of hydrogen and oxygen, which have utility for separating precipitation origin and the effect of evaporation during seasonal periods. The existing isotope record for precipitation and streamflow in the Nile Basin can be used to evaluate predicted streamflow in the Nile River from a changing climate that is expected to induce further changes in precipitation patterns across the Nile Basin.