Geological explorations have revealed plentiful Neogene formation waters in anticlines in the central Qaidam Basin (QB). However, the hydrochemistry and origin of these waters are obscure. In this study, the hydrochemistry and D-O-S-Sr isotopes of these formation waters were determined to study their origin and evolution. The formation waters are enriched in Na-Ca-Cl, and depleted in Mg-K-SO4-HCO3 ions with elevated Li-B-Br-Sr elements. The D-O isotopes prove that the formation waters originated from weak-evaporated meteoric waters, and experienced water-rock interactions. Ion comparisons and Caexcess–Nadeficit diagrams suggest that solute sources of these waters include evaporite dissolution, water-rock interaction, and minor residual lake brines. Bacterial sulfate reduction and water-rock interactions are supported by the high S-Sr isotopes. The enriched Li-B-Br-Sr concentrations of these waters are in accord with the high geochemical background values of the QB. Regarding the genesis of the formation waters, it can be concluded that meteoric waters from the southern Kunlun Mountains were discharged into the basin, weakly evaporated, and then infiltrated into the Neogene strata through faults leaching the soluble ions and mixing with residual lake brines, and all experienced water–rock interactions and a sulphate reduction process.