Abstract: The source region of the Yellow River is a typical alpine and ecologically fragile region in China. Studying spatiotemporal changes in ecosystem services and their trade-off and synergistic relationship is imperative for ecological security and sustainable development in this region. In this study, the InVEST model was used to quantify four ecosystem services (i.e. water yield, soil conservation, carbon storage, and habitat quality) in the source region of the Yellow River from 2000 to 2020. The Spearman correlation coefficient method was used to analyze the relationship between ecosystem services, and the Getis-Ord G_i^* statistical index was used to identify the cold/hot spots of ecosystem services. The trade-offs and synergies of ecosystem services were spatially expressed using the GeoDA software for binary local spatial autocorrelation analysis. The results were as follows. 1) From 2000 to 2020, water yield and soil conservation in the source region of the Yellow River showed a fluctuating trend of “increasing, then decreasing, and then increasing,” while the overall trend was an upward trend with increases of 139.0 mm and 43.20 t·hm⁻², respectively. Carbon storage and habitat quality decreased slightly by 0.4% and 1.9%, respectively. Spatially, water yield and soil conservation exhibited a distribution pattern of “higher in southeast and lower in northwest”. Areas with high carbon storage and habitat quality were predominantly located in the central part of the study area. 2) The hot spots of ecosystem services in the source region of the Yellow River were mainly distributed in the middle of the study area, where forestland and grassland accounted for a high proportion of the land cover. The cold spots were mainly distributed in the source area above Zhaling Lake and Eling Lake, and in the middle northern part of the study area. 3) There was a trade-off relationship between water yield and habitat quality, and the weak trade-off relationship between water yield and carbon storage became a weak synergistic relationship in 2020. There were synergistic relationships between water yield and soil conservation, carbon storage and soil conservation, carbon storage and habitat quality, and soil conservation and habitat quality. Trade-offs between ecosystem services were weakened, and most synergistic relationships were enhanced. 4) In terms of spatial pattern, there was significant spatial heterogeneity of ecosystem service trade-offs and synergies. High-high agglomeration (synergies) were mainly distributed in the central part of the study area, whereas low-low agglomeration (synergies) were mainly located in the western and northern regions. Low-high and high-low agglomeration (trade-offs) were mainly dispersed in the middle northern regions, as well as in the southwestern part of the study area. The results are expected to guide the optimization of ecosystem service spatial patterns and the construction of ecological civilization in the source region of the Yellow River.