Groundwater studies in the South Fork Palouse River Basin have been unable to determine recharge sources, subsystem connectivity and flow patterns due to the discontinuity of pathways in the heterogeneous and anisotropic aquifers located in Columbia River flood basalts and interbedded sediments. Major ion, δ18O, δ2H, δ13C, δ34S and temperature for groundwater collected from 28 wells of varying depths indicate a primary recharge source dominated by snowmelt along the eastern basin margin. This recharge can be separated into two distinct sources—a deeper and relatively less altered snowmelt signal (−17.3‰ to −16.8‰ δ18O, −131‰ to −127‰ δ2H, −12.9‰ to −10‰ δ13C, 18–23 °C) and a more altered signal likely derived from a shallower mixture of snowmelt, precipitation and surface water (−16.1‰ to −15.5‰ δ18O, −121‰ to −117‰ δ2H, −15.9‰ to −12.9‰ δ13C, 12–19 °C). A mixing of the shallow and deep source waters is observed within the upper aquifer of the Grande Ronde Formation near Moscow, Idaho, which results in a homogenization of isotope ratios and geochemistry for groundwater at nearly any depth to the west of this mixing zone. This homogenized signal is prevalent in a likely primary productive zone of an intermediate depth in the overall aquifer system.