Characterization of spatiotemporal variation of the stable isotopes delta O-18 and delta D in surface water is essential to trace the water cycle, indicate moisture sources, and reconstruct paleoaltimetry. In this study, river water, rainwater, and groundwater samples were collected in the Yarlung Tsangpo River (YTR) Basin before (BM) and after the monsoon precipitation (AM) to investigate the delta O-18 and delta D spatiotemporal variation of natural water. Most of the river waters are distributed along GMWL and the line of d-excess = 10 parts per thousand, indicating that they are mainly originated from precipitation. Temporally, the delta O-18 and delta D of river water are higher in BM series (SWL: delta D = 10.26 delta O-18+43.01, R-2 = 0.98) than AM series (SWL: delta D = 9.10 delta O-18 + 26.73, R-2 = 0.82). Spatially, the isotopic compositions of tributaries increase gradually from west to east (BM: delta O-18 = 0.65Lon (degrees)-73.89, R-2 = 0.79; AM: delta O-18 = 0.45Lon (degrees)-57.81, R-2 = 0.70) and from high altitude to low (BM: delta O-18 = -0.0025Alt(m)-73.89, R-2 = 0.66; AM: delta O-18 = -0.0018Alt(m)-10.57, R-2 = 0.58), which conforms to the "continent effect" and "altitude effect" of precipitation. In the lower reaches of the mainstream, rainwater is the main source, so the variations of delta O-18 and delta D are normally elevated with the flow direction. Anomalously, in the middle reaches, the delta O-18(mainstream) and delta D-mainstream values firstly increase and then decrease. From the Saga to Lhaze section, the higher positive values of delta O-18(mainstream) are mainly caused by groundwater afflux, which has high delta O-18 and low d-excess values. The delta O-18(mainstream) decrease from the Lhaze to Qushui section is attributed to the combined action of the import of depleted O-18 and D groundwater and tributaries. Therefore, because of the recharge of groundwater with markedly different delta O-18 and delta D values, the mainstream no longer simply inherits the isotopic composition from precipitation. These results suggest that in the YTR Basin, if the delta O-18 value of surface water is used to trace moisture sources or reconstruct the paleoaltimetry, it is necessary to rule out the influence from groundwater.