Isotopes (δ¹⁸O and δD, δ³⁴S_SO4 and δ¹⁸O_SO4, tritium and ¹⁴C) were employed to reveal moisture sources in precipitation and sources of surface water and groundwater, as well as groundwater residence times and sulfate sources in the Shule River Basin (SRB). Groundwater originates in the Qilian Mountains as high-altitude precipitation and meltwater from ice archives. The local meteoric water line (LMWL) is δD = 7.8δ¹⁸O + 18.1. Precipitation from westerly circulation has a characteristic annual cycle of δ¹⁸O and δD, high (δ¹⁸O > −5‰) in summer and low (δ¹⁸O < −10‰) at other times. This pattern was interrupted by an incursion of the Indian summer monsoon in August 2018, resulting in abnormally low δ¹⁸O and δD values. Surface water in the upper SRB yields an evaporation trend of slope near 5, with an origin near δ¹⁸O = −10‰ on the LMWL. Other catchments of similar altitude in the Qilian Mountains have evaporation trends with different origin points, indicating different input fractions of meltwater from ancient ice for each catchment. Groundwater δ¹⁸O and δD data plot along mixing trends, different in each sub-basin, between three water types: (1) recent Shule River runoff; (2) water like that archived in the Dunde ice sheet, representing precipitation over the last 12 ka; and (3) evaporated water that cannot be explained as precipitation from the last 12 ka. Type (3) water originated as water with δ¹⁸O values between −14 and −20‰ on the LMWL, and may represent incursion of monsoonal circulation prior to 12 ka. Tritium and ¹⁴C data identify post-bomb recharge, but ¹⁴C is of limited use in dating older groundwater mixtures. Sulfate isotopes (δ³⁴S_SO4 and δ¹⁸O_SO4) in dissolved sulfate from groundwater and surface water indicate mixing of sulfur derived from evaporite and sulfide, but do not identify sulfate pollution from fertilizer. Future climate change may lead to water shortage as ancient ice is consumed by melting.