Objective To compare and analyze the genetic variation and differentiation of two tree shrew populations from Guangxi and Yunnan, to promote the breeding of fine strains of tree shrews and optimization of experimental animal models. Methods Sixty-four blood samples were collected and the gene DNA was extracted, 32 from the Guangxi and 32 from Yunnan populations. PCR amplification products of the nine fluorescent labeling simple sequence repeat (SSR) markers were detected by capillary electrophoresis technique and various bioinformatics software were used to analyze the genetic diversity of the two tree shrew populations (Guangxi group and Yunnan group). PCR amplification was performed using 9 fluorescent-labeled simple sequence repeat (SSR) markers, the amplification products were detected by capillary electrophoresis technique, and various bioinformatics softwares such as Popgene were applied to analyze the genetic diversity of the two tree shrew populations. Results The average number of expected heterozygosity and polymorphism information content among the 9 microsatellite loci of Guangxi and Yunnan groups were 0.703 and 0.725, respectively. The mean value of Fis was >0 in both groups. The results of the tests on all the loci with Hardy-Weinberg equilibrium and linkage disequilibrium showed that four loci (CCBL1B, CCDC61, EDA1 and OPA3) in the two populations exhibited significant deviations (P<0.001). The study also found the genetic similarity between the two populations was 0.28, while the genetic distance and unbiased genetic distance were 1.277 and 1.268, respectively. The results from AMOVA analysis showed that 61.57% of genetic variation occurred within populations and 38.43% occurred among populations. Furthermore, structure analysis showed that the Guangxi and Yunnan populations studied in this study are divided into two single subspecies belonging to Tupaia belangeri.Conclusions Our results consistently indicate high genetic diversity of the two tree shrew populations, and their genetic variation is mainly of intra-populational origin.