Abstract: Based on the settlement hierarchy distribution framework of "town-village-historical block", we comprehensively used GIS spatial analysis and geographical detector methods to compare and analyze the spatial characteristics of 421 settlements at different levels in the Grand Canal cultural belt in Jiangsu and Zhejiang Province. We also analyzed the influencing factors from three dimensions of natural geography, human society and canal basin. The results showed that: (1) The distribution of settlements cultural heritage was clustered on the whole, and the smaller the scale of the settlements, the more significant the aggregation of the settlements. However, there was a random or discrete distribution in the city area and the distribution of settlements in each city was uneven. (2) The high-density areas of settlements cultural heritage were located in Hangzhou, Ningbo and Tai Lake area of Suzhou, and there were differences among different types of settlements density areas. (3) The spatial distribution characteristics of settlements cultural heritage were the result of multiple factors, among which the Grand Canal had the greatest influence on the spatial distribution characteristics of settlements. Under the influence of physical geography and canal basin, the overall settlements had low altitude directivity, low slope directivity and adjacent canal directivity, but no obvious exposure directivity. Under the influence of human society factors, the overall settlements tended to be in areas with low traffic accessibility, small population, far away from the city center, and relatively slow economic development. There were differences in various settlements under different influences. (4) Due to the different terrains in the eastern Zhejiang Canal cultural belt, village settlements demonstrated a mountain-oriented, but large-scale settlements were more likely to form near water, rather than near mountain. Based on this, relevant suggestions were put forward to further improve the study of the spatial distribution of settlements cultural heritage in the Grand Canal cultural belt and provide scientific reference and realistic basis for the protection, utilization and inheritance of the Grand Canal culture.