This study is an attempt to understand flow patterns in the Middle Magdalena Valley River basin (MMV), a tropical lowland humid forest. For this purpose, we used δ2H and δ18O water stable isotopes measured in different surface and groundwater sources collected in the area both during the dry and the wet seasons. Two hundred and forty‐five water samples were collected in rainfall, springs, rivers, swamps, and wells (from 10 to 350 m depth). Thus, we were able to establish three tendencies that suggest the existence of surface‐groundwater interactions or preferential groundwater fluxes in the MMV: (i) samples with an isotopic enrichment from the wet to the dry season (groundwater samples following the rainfall isotopic variability), (ii) groundwater samples with an isotopic depletion from the wet to the dry season (opposite to the rainfall isotopic signal), and (iii) groundwater samples whose isotopic composition did not change significantly among seasons. However, none of these tendencies could be related to geology or the type of sampled site, which suggests that some not fully understood processes must be taking place in the hydrological system of the MMV. Both, surface, and groundwater samples are grouped close to the Colombian meteoric line, with exception of some swamp samples collected during the dry period, which displayed the expected evaporation trend line tendency. This may be an indicator of the connection/disconnection of swamps with the river during the hydrological year. Finally, this study aims to fulfil the gap in water‐stable isotope data in Colombia and provide insights about the hydrological setup of a lowland tropical basin.