Abstract: Rural tourism is an important way to promote rural revitalization and achieve common prosperity, and plays an important role in the overall development of urban and rural areas and the coordinated development of regions. Taking the urban agglomeration in the middle reaches of the Yangtze River as the research area, this study systematically analyzed the spatial differentiation of the key rural tourism villages in the urban agglomeration in the middle reaches of the Yangtze River, with the help of ArcGIS 10.6 spatial analysis tool and the methods of nearest proximity index, disequilibrium index and geographic detector. The results showed that: (1) On the whole, the local agglomeration characteristics of rural tourism key villages in the study area were obvious, with 2 high density areas and 4 sub-high density areas, showing the characteristics of being near economically developed, natural and cultural rich areas. They were mainly distributed in the eastern and southern parts, such as Shangrao, Fuzhou, Ji ’an Hengyang, Changsha and other cities. (2) From the perspective of type structure, the rural tourism types of the study area mainly included agricultural production, cultural and folk customs, scenic attraction support, natural ecology and red ruins, and most of them were distributed in a cohesive pattern. The rural tourism key villages with agricultural landscape sightseeing and agricultural product picking as the main products accounted for a large number. The development mode of rural tourism was single; (3) From the perspective of influencing factors, the spatial distribution of the key villages of rural tourism was affected by both natural and human factors, among which the traffic conditions had the most significant influence, followed by permanent population and economic development level. In addition, the spatial distribution of the key villages of rural tourism was the result of the comprehensive effects of natural and social economic factors. The effect of the interaction of two factors was more significant than that of the single factor, showing two kinds of effects: nonlinear enhancement and double enhancement. Finally, according to the type structure and spatial distribution of typical rural tourism resources , this study provided suggestions on the linkage development of rural tourism, in order to strengthen the role of rural tourism in urban and rural coordination and regional development.