As important nodes of the 21st-Century Maritime Silk Road, ports and port cities play the key role in promoting the development of countries along the road. Based on Google Earth and Landsat remote sensing data and the container throughput statistics, we adopt the spatial analysis method to analyze the temporal and spatial variation characteristics of 38 important ports in the countries (excluding China) along the 21st-Century Maritime Silk Road. Furthermore, we introduce the urban construction land area as a representative parameter of urban development to modify the relative concentration index (RCI), and evaluate the relationships between ports and port cities along the road. The results are shown as follows. 1) The areas of important ports along the 21st-Century Maritime Silk Road have obvious regional distribution patterns, showing the characteristic of "bigger in the west and smaller in the east". 2) The variation of the port area (0.01-4.93 km²) and the change proportion of the port area (1%-507%) have the characteristics of regional differentiation. In the different regions (Southeast Asia, South Asia, West Asia, Africa, and Europe), the differences in port area change are little, while the differences in port area change proportion are large. 3) The port-city relationships also have obvious regional features. The influences of European ports on the cities are relatively strong (average RCI is about 2.98 in 2017), and the role of the ports in Asia is relatively weak (average RCI is about 1.51 in 2017). The results provide scientific information for construction of ports and port hinterlands.