Karst springs of the Zagros Mountains contribute a significant amount to agricultural and human water demands of western and south-western Iran. For an adequate management of available water resources in semi-arid and arid regions, sufficient hydrological monitoring is needed and hydro-chemical and isotope hydrological data provide important additional information. About 350 water samples were collected from precipitation, river water, and karst springs of the upper part of the Karkheh River Basin (20,895 km2) located between 33º 35´ and 34º 55´ North and 46º 22´ and 49º 10´ East with elevations ranging from 928 to 3563 m above sea level. Sampling was conducted in monthly time resolution from August 2011to July 2012. All samples were analyzed for hydro-chemical parameters (pH, electrical conductivity, and major ions) and stable isotopes (deuterium, oxygen-18). Isotope values of precipitation indicate a local meteoric water line (Zagros MWL δ2H = 6.8 δ18O + 10.1; R2 = 0.99) situated between the Mediterranean MWL and Global MWL. Spring and river water isotope values vary between −7.1 and −4.1%, and −38 and −25% for δ18O and δ2H, respectively, responding to winter snowmelt and evaporation. This work implements stable isotopes and hydro-chemical information of springs and river water to understand hydrological and hydro-geological interrelations in karstic semi-arid areas and helps to improve the current water resources management practices of western Iran.