The role of groundwater in maintaining streamflow in the alpine area with distribution of permafrost and seasonal frost is a poorly studied topic of considerable interest. The stream and groundwater interactions and groundwater contributions to the Heihe River were investigated during this study in a representative subcatchment in the headwater region of the Heihe River Basin, the northeastern Qinghai-Tibet Plateau of China. The hydraulic, chemical and isotopic data as well as Bayesian mixing model results show that groundwater-stream water interactions were both spatially and temporally variable. The tributaries were primarily recharged by springs within the permafrost zone during the frozen period when the water source and sediments were frozen and the groundwater discharged to the mainstream within the seasonal frost zone to maintain the streamflow. The groundwater contribution to mainstream discharge decreased from 95% during the frozen period to 80-90% in the thawing period due to the inflow from tributaries. However, the stream and groundwater interactions vary several times along altitude during the thawed period from June to early September, due to the increased glacial/snow meltwater volume, deepened active layer, and melted seasonal frost. Groundwater contribution decreased to ~40-60% of the mainstream discharge during the thawed period because tributary streams contribution largely increased. As shown by ~70-90% contribution from groundwater to the mainstream discharge, the mainstream flow mainly sourced from the release of groundwater in aquifers in the freeze-back period. These data indicate that the variations in groundwater-surface water interactions were largely influenced by the distribution and freeze-thaw cycle of permafrost and seasonal frost. The importance of groundwater storage in maintaining streamflow in the Heihe headwater region was highlighted by this study.