【Objective】The conservation of plant species with limited distribution and endangered status is a critical concern in biodiversity preservation. Among such species, Pseudotaxus chienii, a nationally protected plant species, has attracted significant attention due to its narrow habitat range and its vulnerability to extinction. As an endemic species primarily found in the mountainous regions of Zhejiang and Jiangxi provinces in China, P. chienii plays an important ecological role in maintaining forest biodiversity and stabilizing the local ecosystem. Unfortunately, over the past few decades, the natural populations of P. chienii have been severely affected by habitat destruction, fragmentation, and anthropogenic pressures, such as overharvesting and deforestation. These factors have contributed to a significant decline in both the population size and genetic diversity of this species. The research also sought to provide a deeper understanding of the evolutionary processes shaping the genetic makeup of P. chienii, especially in light of its restricted geographic range. By examining the genetic variation within and among populations, this study aimed to determine whether there are significant genetic differences that could have implications for long-term species survival. Additionally, the study aimed to investigate the extent of inbreeding and heterozygosity levels within these populations, which are important indicators of the overall fitness and adaptability of the species. The ultimate goal of the research was to generate critical genetic data that would serve as a foundation for future conservation and population restoration efforts, enabling the development of strategies that could enhance the species' genetic diversity and resilience in the wild.【Method】To achieve these objectives, a total of 133 individuals were collected from six natural populations of P. chienii located in different regions of Zhejiang Province (Fengyang Mountain Nature Reserve) and Quzhou City (Tianji Longmen Mountain Range). These populations were chosen for their geographic diversity, allowing for an examination of genetic variation across a range of environmental conditions and altitudes. In order to assess the genetic diversity and population structure of P. chienii, simple sequence repeat (SSR) molecular markers were employed. SSR markers are highly effective in detecting genetic variation and are widely used in plant population genetics studies. A total of 14 SSR primer pairs, known for their high polymorphism and ability to amplify variable loci, were selected for genotyping the 133 individuals. These markers were used to analyze a wide range of genetic parameters, including the number of alleles, the effective number of alleles (N_e), the Shannon's diversity index (I), and polymorphic information content (PIC). These parameters are commonly used to assess the level of genetic diversity within populations. Additionally, observed and expected heterozygosity (H_o and H_e) were calculated to evaluate the extent of genetic variation at the individual level, while fixation coefficients (F) and inbreeding coefficients (F_is) were determined to identify potential inbreeding and genetic drift within the populations. Furthermore, to explore the genetic structure of P. chienii, both principal coordinate analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA) clustering were applied. These multivariate statistical methods allowed for the visualization of genetic relationships among individuals and provided insights into the clustering patterns of populations based on genetic similarities. By conducting these analyses, the study aimed to reveal any genetic subgroups within the species and identify the factors that contribute to the observed genetic structure. In summary, this study used advanced molecular techniques to investigate the genetic diversity and structure of P. chienii populations. By analyzing a large number of individuals from multiple natural populations, the study aimed to provide a comprehensive picture of the genetic variation within the species, and to understand the evolutionary and ecological factors that contribute to this variation.【Result】Significant differences were observed in tree height and diameter at breast height among the six populations of P. chienii, with the FYS2 population exhibiting the best growth status. Using 14 pairs of SSR primers with good polymorphism, genotyping of 133 P. chienii individuals identified a total of 102 alleles. The mean values of effective number of alleles (N_e), Shannon's diversity index (I), and polymorphic information content (PIC) were 4.69, 1.61 and 0.86, respectively. The average values of observed heterozygosity (H_o), expected heterozygosity (H_e), inbreeding coefficient (F_is) and fixation coefficient (F) were 0.34, 0.73, 0.54 and 0.58, respectively. Overall, the results indicated high genetic diversity in P. chienii populations, but there was a moderate level of heterozygote deficiency and obvious inbreeding within populations. Genetic structure analysis divided P. chienii individuals into two subgroups, which was also supported by PCoA and UPGMA clustering analysis.【Conclusion】The natural populations of P. chienii exhibit high levels of genetic diversity, with significant differences in genetic diversity among populations. Proximity-based clustering analysis reveals that geographically adjacent natural populations of P. chienii tend to cluster into the same subgroups, indicating that geographic variation is the primary factor influencing differences in the genetic structure in P. chienii.