Abstract: Quasipaa spinosa is a large-scale frog species with a unique distribution in Fujian Province and is listed as a protected species under the “Three Protected Categories”. The development of new rapid methods to identify different geographical populations of Quasipaa spinosa in Fujian Province is extremely important for the protection of this species. In this study, the DNA was extracted from the wild Quasipaa spinosa samples collected from 14 regions in Fujian Province, and the mitochondrial 12S rRNA gene was amplified by using PCR. Then, the first-generation sequencing was conducted. The evolutionary tree was constructed based on the obtained 12S rRNA gene sequence. The results showed that Quasipaa spinosa in Fujian Province could be divided into two major populations: Wuyishan, Jianyang, Shunchang, Jiangle, Mingxi, Yong'an, Hua'an, Dehua and Xinluo were clustered into one group (the western population). Wuyishan, Zhenghe, Pingnan, Ningde, Fuding, Yongtai, Dehua, Xinluo and Fu'an were clustered into another group (the eastern population). Among them, Wuyishan, Dehua and Xinluo were distributed in both major categories. In order to distinguish the two populations based on the 12S rRNA gene more quickly, the SNP loci of the 12S rRNA gene in the two major populations were screened. And a rapid specific SNP genotyping method for these two populations was developed based on the competitive allele-specific PCR (KASP, Kompetitive Allele-Specific PCR) method. The results showed that the developed KASP detection system could accurately distinguish the two lineages of wild Quasipaa spinosa in Fujian Province. The results could provide a scientific basis for the in-depth understanding of the population differentiation of Quasipaa spinosa in Fujian Province, and the effective identification of different geographical populations, as well as the protection and breeding of germplasm resources.