High glacial mountain environments around the world are important water reservoirs and are extremely sensitive to the effects of climate change. Considering that climate change affects snow accumulation and glacial melting, it is essential to understand the runoff generation and hydrological dynamics in these type of systems. Andean glaciers have shown a retreating and thinning pattern since the start of the 21st century. Regarding this, the stable isotopic composition of waters is useful to assess the contributions from different sources (i.e., glaciers, precipitation, and tributaries' inputs) to rivers. Several Patagonian mountain rivers have Andean glaciers in their headwaters, one of which is the Manso River. In this basin, over the last 30 years, the Ventisquero Negro Glacier (Manso Glacier's regenerated tongue) experienced a rapid thinning and recession that generated a proglacial lake, called Manso proglacial Lake. In this study, topographic effects upon the stable isotopic composition of river waters (δ2H and δ18O) are recognized, particularly, the altitude effect. The stable isotope composition of the Manso River varies downstream, evolving to a composition more enriched in heavy isotopes in the lower basin, likely due to altitude effects and the progressive evaporation that mainly occurs in the lakes' surfaces that the river crosses. Results show that glacial meltwater is the major water contributor in the upper basin during early fall. In the rest of the basin the stable isotopic composition of the Manso River is mainly controlled by the lakes' isotopic signatures and is highly influenced by two tributary rivers. The concordance between isotopic models and hydrological data demonstrates that stable isotopes constitute a valuable tool to quantify the contribution of different water sources. This has the potential to be particularly useful in areas where discharge data is unavailable.