The Colombian Andean Mountains host the headwaters of the main basins of the country. However, the interactions between high-mountain ecosystems and the isotopic composition of water in this region has been poorly studied. Here we present and analyze the first set of stable isotopes data collected in the Central Andes of Colombia. Stable isotopic 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 δ ²H, δ ¹⁸O and d-excess were identified and compared to daily air temperature and precipitation at seven meteorological stations. A Local Meteoric Water Line was defined as δ²H = 8.13 δ ¹⁸O + 12.5, R ²=0.98. δ ²H, δ ¹⁸O and d-excess values of precipitation were more negative during the rainy season and changes were more related to precipitation events and amounts than to temperature. An altitude effect of -0.11 ‰ / 100 m and -0.18 ‰ / 100 m was estimated for stream water and precipitation, respectively, where the latter showed a non-linear behavior. The data set was compared to stations of the Global Network of Isotopes in Precipitation (GNIP) database in Colombia and a back-trajectory analysis of air masses was conducted to compare with d-excess. δ ¹⁸O weighted means changed with respect to the position of the Central Andes and the altitudinal range 2,100 to 3,100 m a.s.l.. High d-excess can be attributed to moisture recycling enhanced by the local ecosystems and the travel of precipitable water from the Amazon basin across the northern Andes. The results showed a high range of variation due to the differences in elevation, seasonality and atmospheric circulation patterns across the year. The present study contributes to fill the gap of spatial and temporal isotopic composition data in the northern Andes as well as to the implementation of the first “National Network for Isotopes” in Colombia.