Abstract: Quasipaa spinosa, a large frog species with a unique distribution in Fujian Province and listed as a protected species under the “Three Protected Categories,” is of great importance for conservation. Developing new rapid methods to distinguish different geographical populations of Q. spinosa within Fujian Province is crucial for the protection of this species. In this study, wild Q. spinosa samples were collected from 14 regions in Fujian Province. DNA was extracted, and the mitochondrial 12S rRNA gene was amplified using PCR and then sequenced using Sanger sequencing. An evolutionary tree was constructed based on the obtained 12S rRNA gene sequences of wild Q. spinosa from the 14 regions, revealing that the Q. spinosa in Fujian Province can be divided into two major populations: one group includes Wuyi Mountain, Jianyang, Shunchang, Jiangle, Mingxi, Yongan, Hua'an, Dehua, and Xinluo（the western population), while the other group includes Wuyi Mountain, Zhenghe, Pingnan, Ningde, Fuding, Yongtai, Dehua, and Xinluo（the eastern population). Among them, Wuyi Mountain, Dehua, and Xinluo are distributed in both major groups. To further rapidly distinguish between the two populations based on the 12S rRNA gene, we screened the SNP loci of the 12S rRNA gene in these two geographical populations and developed a rapid SNP analysis method based on the competitive allele-specific PCR（KASP）technique. The results showed that the developed KASP molecular markers can accurately distinguish between the two lineages of wild Q. spinosa in Fujian Province. This study provides a scientific basis for understanding the population differentiation of Q. spinosa within Fujian province, identifying different geographical populations, and conserving germplasm resources and breeding.