Abstract: China is one of the countries with the richest diversity of giant tarantula species, with a total of 30 species in 2 genera recorded so far. Yunnan Province possesses more than half of the giant tarantula species resources in China. However, research on the genetic differentiation of giant tarantula populations in different geographic regions of Yunnan Province has been relatively limited. By studying the degree of genetic differentiation among giant tarantula populations from different geographic regions in Yunnan Province, we have uncovered the molecular biological mechanisms underlying the ongoing evolution of giant tarantulas in this region. We collected giant tarantula samples from six prefectures in Yunnan Province and conducted genetic differentiation analysis among the populations based on the determined COⅠ and 18S rDNA gene sequences. Through sequence variation and phylogenetic tree analysis, we investigated the genetic distances, molecular phylogenetic relationships, and degree of genetic differentiation among giant tarantula populations in different geographic regions of Yunnan Province. The experimental results indicated that there were 342 and 241 variable sites (accounting for 74.02% and 55.52% of the total sequence length) in the COⅠ and 18S rDNA gene sequences of the six geographic populations of giant tarantulas, respectively, and the average nucleotide divergence (K) of the total population was 157.553 and 94.287, respectively, indicating a high level of genetic diversity in giant tarantulas. The average values of Tajima's D and Fu's F_s for the total population were both positive, suggesting that the evolution of the total giant tarantula population is under balancing selection, and the genetic differentiation is not significant. The average genetic distances between different geographic populations were 0.586 and 0.437, and the Mantel test based on geographic distance and genetic distance showed no significant correlation between them. Based on the phylogenetic trees constructed for each geographic population, and the clustering results of the sequences, no clear geographic lineages were found.