Abstract: The Yellow River Basin bears the heavy responsibility of national energy and food production with scarce water resources. Under the background of limited resources yet ever-expanding demand, we explored the pressure and challenges faced by the sustainable development of the Yellow River Basin, which are brought upon by the spatial mismatch of water-energy-food resources, in the hope of providing a scientific basis and reference for the high-quality development of the basin, as well as the national and regional coordinated development strategy. Based on the mesoscale perspective, the comprehensive evaluation framework of water-energy-food system in the prefecture-level cities in the Yellow River Basin is constructed to analyze the coupling and coordinated development level and spatial correlation characteristics of water-energy-food system in prefecture level cities in the Yellow River Basin. The results show that from 2000 to 2018, the coupling coordination degree of water-energy-food system of the prefecture-level cities in the basin evolved from primary coordination to intermediate coordination, but the coordinated development was insufficiently unstable. By 2018, the degree of system coupling and coordination showed a downward trend. In most years, the system coupling coordination degree showed significant spatial correlation characteristics. From the perspective of the prefecture-level cities, the research on water-energy-food system is more regionally targeted, and it is easier to highlight the individual characteristics of system development at the mesoscale. Further study should be conducted on the influencing mechanism of the spatial correlation of water-energy-food system, and the deconstruction of the dynamic coupling and coordinated development mode of this system in the Yellow River Basin.