Abstract: The analysis of spatial differentiation and it’s influence factors of traditional villages can provide theoretical support for the overall protection and zoning protection of traditional villages, and also provide a scientific basis for formulating protection policies. Taking 550 traditional villages in Hunan, Hubei and Jiangxi Provinces as research objects, this paper use the methods of spatial analysis and mathematical statistics to analyse the spatial differentiation and it’s influence factors of traditional villages. The research is carried out from two scales of prefecture and county. The results showed that the traditional villages in Hunan, Hubei and Jiangxi Provinces are characterized by widespread distribution and partial concentration. The distribution pattern is a type of agglomeration and the agglomeration characteristics of county scale are more obvious than prefecture scale. The plain of Jianghan-Dongting lake is a low-low agglomeration district and the distribution of traditional villages is sparse. The number of traditional villages is positively related to the average elevation and average annual rainfall. It is negatively correlated with river density, average annual temperature, highway density, urbanization rate, per capita GDP and population density. The average elevation, highway density have a significant impact on the distribution of traditional villages at the prefecture scale. The average annual temperature, average annual rainfall and per capita GDP have a significant impact on the distribution of traditional villages at the county scale. The natural geographical factors are the environmental basis for the formation of traditional villages, and affect the preservation of traditional villages by affecting social and economic activities in Hunan, Hubei and Jiangxi Provinces.