The Colorado River has been identified as the most overallocated river in the world. Considering predicted future imbalances between water supply and demand and the growing recognition that base flow (a proxy for groundwater discharge to streams) is critical for sustaining flow in streams and rivers, there is a need to develop methods to better quantify present-day base flow across large regions. We adapted and applied the spatially referenced regression on watershed attributes (SPARROW) water quality model to assess the spatial distribution of base flow, the fraction of streamflow supported by base flow, and estimates of and potential processes contributing to the amount of base flow that is lost during in-stream transport in the Upper Colorado River Basin (UCRB). On average, 56% of the streamflow in the UCRB originated as base flow, and precipitation was identified as the dominant driver of spatial variability in base flow at the scale of the UCRB, with the majority of base flow discharge to streams occurring in upper elevation watersheds. The model estimates an average of 1.8 × 1010 m3/yr of base flow in the UCRB; greater than 80% of which is lost during in-stream transport to the Lower Colorado River Basin via processes including evapotranspiration and water diversion for irrigation. Our results indicate that surface waters in the Colorado River Basin are dependent on base flow, and that management approaches that consider groundwater and surface water as a joint resource will be needed to effectively manage current and future water resources in the Basin.