Abstract: The space genes of settlements, which were carried and contained affluent cultural connotations, and along with the exchange, dissemination and intermingling of ethnic cultures, subsequently, feedback regulation of the space genes will come into being and the phenomenon of space genetic derivation of settlements. The ethnic mixed settlements, which consisted of the descendants of the Han nationality of Tunpu and the ethnic minorities, is a special kind of traditional settlement in Guizhou, which were the products of mutual contact, borrowing, fusion, and accumulation of different ethnic groups in a specific historical and geographical environment. From the perspective of ethnic cultural integration, the study takes Zhenshan Village, a typical village inhabited by the descendants of the Tunpu and the Buyi ethnic groups, as an example, and integrates the connotation and functional mechanism of the space gene, and utilizes relevant research methods such as architecture and cultural anthropology to compare and analyze the space gene of the settlements in the Shangzhai and Xiazhai of Zhenshan villages. The results show that there are a lot of derivative phenomena in the space genes of the overall layout, the structure of street, places, courtyards, and architectural decorations in the Shangzhai and Xiazhai of Zhenshan villages, which were triggered by the fusion of ethnic cultures, and they are generated by the four external thrusts of the same social background conditions, mixed and coexisting living environment, accept each other in the interracial marriage, and interconnected national cultural beliefs. It helps to understand the formation and space evolution patterns of similar ethnically mixed settlements that are widely found in Guizhou.