In order to understand the present situation of germplasm resources of different Cuora flavomarginata in Zhejiang Province, the genetic diversity and genetic structure of eight C. flavomarginata populations from wild and breeding farms in Zhejiang Province were compared based on mitochondrial DNA COI sequence. The results showed that the se⁃ quence length of COI gene of C. flavomarginata was 1,040 bp, and the average base content was A=27. 4%、T=30. 5%、C= 24. 6%、G=17. 5%, with strong AT preference. Nine haplotypes were identified in the eight populations, and the haplotype diversity (Hd) was (0. 79900±0. 21000), and the nucleotide diversity (Pi) was (0. 00209±0. 00147), showing“ high Hd and low Pi” genetic distribution. The genetic distance between different populations ranged from 0 to 0. 003. The genetic distance within populations was between 0 and 0. 004, among which the genetic distance within Pan’an Houge Village (YS) population was the largest, which was 0. 004. The haplotype phylogenetic tree did not show obvious pedigree structure. The genetic differentiation index (FST) interval was from -0. 34752 to 0. 82222, and the genetic differentiation was not significant (p>0. 05) in eight populations. Tajima’s D and Fu’s FS test values were either positive (Tajima’s D=-0. 36627, Fu’s FS=-0. 605), but statistically insignificant. The distribution of nucleotide mismatches indicated that the population of the C. flavomarginata in Zhejiang Province did not experience a population expansion event. Mantel test showed that there was no significant correlation between genetic distance and geographical distance (R=0. 2938,p>0. 05). AMOVA analysis found that the genetic variation within populations was higher than that between populations (85. 06%>14. 94%). The results showed that the populations of C. flavomarginata in Zhejiang Province are characterized by high haplotype diversity and low nucleic acid genetic diversity, and there was a certain level of genetic differentiation, and there was obvious gene exchange among the populations. The wild C. flavomarginata obtained in Pan’an County was identified as Zhejiang species according to COI gene analysis. Moreover, it was also found that there were hybrids between Zhejiang species and Anhui species. It is suggested that the C. flavomarginata populations with different degrees of differentiation should be divided into different units for protection. This study uses genetic diversity analysis to provide a basis for the development and utilization of C. flavomarginata germplasm resources in Zhejiang Province and the formulation of reasonable protection measures. [ABSTRACT FROM AUTHOR]