The chemical and isotopic (87Sr/86Sr, δ11B, δ34Ssulfate, δ18Owater, δ15Nnitrate) compositions of water from the Lower Jordan River and its major tributaries between the Sea of Galilee and the Dead Sea were determined in order to reveal the origin of the salinity of the Jordan River. We identified three separate hydrological zones along the flow of the river: (1) A northern section (20 km downstream of its source) where the base flow composed of diverted saline and wastewaters is modified due to discharge of shallow sulfate-rich groundwater, characterized by low 87Sr/86Sr (0.7072), δ34Ssulfate (-2‰), high δ11B (∼36‰), δ15Nnitrate (∼15‰) and high δ18Owater (-2 to-3‰) values. The shallow groundwater is derived from agricultural drainage water mixed with natural saline groundwater and discharges to both the Jordan and Yarmouk rivers. The contribution of the groundwater component in the Jordan River flow, deduced from mixing relationships of solutes and strontium isotopes, varies from 20 to 50% of the total flow. (2) A central zone (20-50 km downstream from its source) where salt variations are minimal and the rise of 87Sr/86Sr and SO4/Cl ratios reflects predominance of eastern surface water flows. (3) A southern section (50-100 km downstream of its source) where the total dissolved solids of the Jordan River increase, particularly during the spring (70-80 km) and summer (80-100 km) to values as high as 11.1 g/L. Variations in the chemical and isotopic compositions of river water along the southern section suggest that the Zarqa River (87Sr/86Sr∼0.70865; δ11B∼25‰) has a negligible affect on the Jordan River. Instead, the river quality is influenced primarily by groundwater discharge composed of sulfate-rich saline groundwater (Cl-=31-180 mM; SO4/Cl∼0.2-0.5; Br/Cl∼2-3×10-3; 87Sr/86Sr∼0.70805; δ11B∼30‰; δ15Nnitrate ∼17‰, δ34Ssulfate=4-10‰), and Ca-chloride Rift valley brines (Cl-=846-1500 mM; Br/Cl∼6-8×10-3; 87Sr/86Sr∼0.7080; δ11B∼40‰; δ34Ssulfate=4-10‰). Mixing calculations indicate that the groundwater discharged to the river is composed of varying proportions of brines and sulfate-rich saline groundwater. Solute mass balance calculations point to a ∼10% contribution of saline groundwater (Cl-=282 to 564 mM) to the river. A high nitrate level (up to 2.5 mM) in the groundwater suggests that drainage of wastewater derived irrigation water is an important source for the groundwater. This irrigation water appears to leach Pleistocene sediments of the Jordan Valley resulting in elevated sulfate contents and altered strontium and boron isotopic compositions of the groundwater that in turn impacts the water quality of the lower Jordan River.