Abstract: Influenced by the natural geographical environment and the multi-ethnic culture, the rural settlement clusters along the Gejiu-Bisezhai-Shiping Railway in southern Yunnan have formed a unique spatial form during the adaptation process to railway technology, natural terrain and regional culture. This form is characterized by 'the settlement area as the core, strip growth, and layered symbiosis', reflecting the complex interaction logic of space, nature and culture. Guided by the 'spatial gene' theory and based on the framework of 'macro landscapemedium-sized settlement-micro architecture', this paper summarizes the spatial gene characteristics of the rural settlement clusters along the Gejiu-Bisezhai-Shiping Railway in the section from Jijie to Shiping in terms of site selection pattern, settlement distribution, spatial form and architectural form, and analyzes the mechanism of the formation and development of spatial genes from the perspectives of natural geographical constraints, ethnic cultural integration, social and economic changes, and construction technology inheritance. The research aims to identify and explain the spatial genetic code of this special type of settlement, with the intention of providing theoretical basis and practical references for the systematic protection of the railway heritage in southern Yunnan and the organic renewal of rural areas.