With the increasingly serious water shortage and other problems, Inter-basin water transfer (IBWT) has become an important measure to alleviate regional water stress. In this study, based on the improved water stress index (WSI), we comprehensively assessed the multi-scale (urban, tertiary basin zones) spatio-temporal status of WSI in the four major basins of the Huang, Huai, Hai and Yangtze River Basins (HHHYRB) from 1965 to 2020, and analysed the contribution of Inter-basin water transfer to alleviate regional water stress. The Dagum Gini coefficient method was used to determine the differences and sources of WSI distribution. Finally, the impact of each water transfer project was quantitatively analysed through bottom-up scenario derivation. The results show that 35.8% of the cities in the HHHYRB, and 40.4% of the tertiary basins are at medium or higher risk of water stress. The impacts of external inter-basin water transfers in the HHHYRB are all less than 0.002. The IBWT effectively mitigated the WSI in the Hai River Basin (75%) and the Huaihe River Basin (15.6%), negatively affected the WSI in the Yellow River Basin (−6.4%), and had only a 2% impact on the Yangtze River Basin. There is obvious spatial heterogeneity in the WSI of the HHHYRB, and the coefficients between groups (48.3%–66%) are higher than the coefficients within groups (17.3%–23.7%) and hypervariable density coefficients (10.8%–31%). IBWT projects effectively moderate the degree of inequality in water resources, with intra-basin impacts ranging from 1.77% to 33.69% and inter-basin impacts reaching 2.29%–7.28%. Most of the IBWT projects tend to transfer water from areas with low WSI to areas with high WSI with positive impacts, but there are still 10 water transfer projects with negative impacts. Therefore, the impacts of IBWT should be considered comprehensively when formulating water resources management policies in order to achieve long-term sustainable use of water resources.