AbstractThe Colombian Andean Mountains include the headwaters of the main basins of the country. However, the isotope composition of water in these high mountain ecosystems has been poorly studied. In this study, we analysed the first set of stable isotope data collected along a wide elevation range (2600–4950 m a.s.l.) in the Central Andes of Colombia. The stable isotope composition of stream water and precipitation was determined for a period between 2017 and 2018 in the Upper Claro River basin. The driving factors influencing the spatial and temporal variability of δ²H, δ¹⁸O, and d‐excess were identified, and compared with daily air temperature and precipitation data from seven meteorological stations. The local regression line was described by δ²H = 8.2 δ¹⁸O + 12.3, R² = 0.98. The δ²H and δ¹⁸O values showed more depletion in heavy isotopes, and the d‐excess values were more negative during the rainy season. An altitude effect of −0.11‰/100 m and −0.18‰/100 m was estimated for stream water and precipitation δ¹⁸O values, respectively, with the latter showing non‐linear behaviour. The dataset was compared with Colombian stations of the Global Network of Isotopes in Precipitation database, and a back‐trajectory analysis of air masses was conducted and compared with the d‐excess values. The δ¹⁸O weighted mean values changed with respect to the position in the Central Andes, indicating contrasting altitude effects depending on the moisture sources. The most positive d‐excess values were attributed to moisture recycling enhanced by local ecosystem conditions and the origin of precipitation from the Amazon basin, which change during the year and across the northern Andes. The results showed a high level of variation because of differences in elevation, seasonality, and atmospheric circulation patterns during the year. This study contributes to knowledge of spatial and temporal isotope composition data in the northern Andes, delineation of water supply basins, and to the definition of ecosystem boundaries in the high mountains of Colombia.