Reference evapotranspiration (ETo) is the basic component of the hydrological cycle. This study systematically analyzed the spatiotemporal variations of ETo in the Three-River Headwaters Region (TRHR) by utilizing the FAO Penman-Monteith equation during the period of 1961-2016, and studied the impact of climatic variables on ETo fluctuation based on sensitivity analysis and contribution analysis. Over the past 56 years, ETo increased in most months and annually in most regions of the TRHR, and the spatial distribution for annual ETo varied apparently. Sensitivity analysis revealed that ETo responded more to changes in maximum temperature and sunshine duration than relative humidity, wind speed, and minimum temperature. The relative importance of climatic variables to ETo was influenced by the ETo sensitivity and the fluctuation of climatic variables. Contribution analysis indicated that the significant temperature increase was mainly responsible for the observed increase in ETo. Besides, spatially distributed analysis showed that the annual TRHR ETo tended to increase from east to west, north to south, and high to low elevation. The analysis also indicated a dominant altitudinal spatial gradient in the sensitivity of ETo to changes in different climatic variables. The physical mechanisms for ETo variations discussed in this study might provide a reference for water resources management and conservation of high-elevation areas.