Abstract: Influenced by the physical geographical environment and national cultural characteristics, Miao settlements in mountainous areas of western Hunan gradually grow into compact, three-dimensional, and nationalized in the process of adapting to the landscape environment and regional culture, which reflects the good logical relationship between space and nature, space and history. Under the guidance of ‘space gene’ theory, the paper constructs a three-level space gene identification and analysis framework of ‘macro landscape-meso settlementmicro architecture’, summarizes the space gene characteristics and discusses the generation mechanism of Miao settlements in mountainous areas of western Hunan. It is a useful practice under the guidance of space gene theory, aiming at protecting the diversity of traditional regional culture and providing a scientific basis for the protection and inheritance of ‘local characteristics’ of characteristic rural settlements.