Stable O and H isotope data distinguish three sources for base flow in five reaches of the San Pedro River: (A) base flow and sub-flow from upstream reaches of the river; (B) bank storage derived from summer monsoon floodwater; and (C) water from the mountainous flanks of the river catchment. A and C support base flow in the sub-basin upstream of Sierra Vista. A, B, and C combine to support base flow near St. David. Source C in this area is ancient deep-basin groundwater. Source C dominates in Cascabel near Benson Narrows, with downstream additions from A. In Cascabel near Gamez Road, sources A and C combined to support base flow that had disappeared by 2019. Near Redington, source C appears to have operated through a limestone aquifer vulnerable to short-term drought. Groundwater sub-basins separated by impermeable sills in the riverbed are evolving into hydrologically separate sub-basins as base flow across the sills decreases. The decrease in base flow partly reflects regional long-term drought, which has been exacerbated by pumping. Additional groundwater demand from urban growth upstream of Benson is likely to cause further decline of base flow near St. David and Sierra Vista.