Tropical mountain regions are characterized by strong spatial climate gradients which together with the limited amount of data and knowledge of the underlying processes hinder the management of the water resources. Especially for regional-scale prediction it is important to identify the dominant factors controlling the rainfall–runoff response and link those to known spatial patterns of climate, soils, and vegetation. This study analyzes the rainfall–runoff relation of 13 intensively monitored micro-catchments in the Andes of southern Ecuador. The results of this study show that streamflow in the southern cordillera of the Ecuadorian Andes, above 2500 m a.s.l., primarily consists of subsurface flow. The yearly amount of streamflow is controlled by the annual rainfall depth, whereas the temporal distribution is mainly governed by the lateral saturated hydraulic conductivity, the soil water retention and the antecedent soil moisture content. Anthropogenic effects were found insignificant, with the exception in one of the studied micro-catchment. Effect of land use changes in most of the micro-catchments did not reflect in the shape of the flow duration curve because either the spatial extent of human impact was small and/or the overall basin slope was less than 20%.