The Gorge of the Yarmouk River is the drainage base for groundwater flowing from different natural replenishment areas and aquifer lithologies. For the three riparian states (Syria, Jordan and Israel), the uncertainty of groundwater origin imposes difficulties on the management of the water flowing in the Basin towards the Gorge. In the Yarmouk basin, groundwater replenishment areas are flanks of Mt. Hermon, the Golan Heights, Jebel Druze and the Ajloun Dome. The catchment lithology of these areas is mostly alkali olivine basalts and partly limestones. Most of the groundwater accumulates in Upper Cretaceous limestone aquifers (A7/B2) underlying the basalts. The chemistry of groundwater flowing from the Golan Heights and from Jebel Druze indicates origin from basaltic rocks. In the Ajloun, the chemistry indicates mixing of water from basalts and limestones with local dissolution of gypsum and halite. The Cretaceous outcrops in the southern and southeastern flanks of the Palmyra structure are the origin of the northern contributors of groundwater. The Mg2+/Ca2+ values of groundwater from the Hauran, Golan and Ajloun regions scatter between 1.17 and 0.54. Samples from the Ajloun and Golan are characterized by very low values of 0.54–0.61 indicating significant dissolution of gypsum/anhydrite. Albitization of plagioclase from basalts is observed in the Golan region by decrease of Na+/Cl− and corresponding increase of Ca2+/Cl−. Correlation plots between various ions and ionic ratios are presented to differentiate between the different sources of groundwater emerging in the Lower Yarmouk Gorge (LYG) which is an important step for responsible management of transboundary water resources in this region. The presented methodology was found by the authors as very useful for identifying different groundwater bodies in multi-aquifer systems by Möller et al. (J Hydrol Reg Stud 5:33–47, 2016).