High permeability karstic aquifers and low recharge rates in arid and semi-arid regions lead to the formation of extensive (often interbasin) regional flow systems such as are documented in Cuatrociénegas valley. The flow system discharges to springs that support unique spring-fed ecosystems. The springs occur in lines parallel to the Sierra San Marcos that divides the valley into west and east sub-basins, The west sub-basin hosts fracture-controlled springs with relatively high and constant discharge, elevated temperatures, and high conductivity. The east sub-basin is comprised of dozens of stratigraphically-controlled small springs adjacent to alluvial fans with lower temperature, lower conductivity, and relatively low discharge affected by precipitation. Isotopic data (tritium, 2H/18O, and 3H/3Hetrit) from the springs show that regional groundwater flow predominates with local recharge also occurring in the mountains surrounding Cuatrociénegas and also that mountain recharge older than 50 years predominates. Analysis of permeability and recharge data predicts recent interbasin flow from the Ocampo Valley to the north and the Hundido Valley to the southwest and perhaps farther. Limited by sparse data, research on quantification of the flow system in this karstic regional aquifer system and the precise locations and rates of local recharge is required to preserve spring flows, which are also threatened by climate change, population growth, and aquifer overpumping. This will require more water wells, pump tests, and water quality data on local and regional scales.