Abstract: The genetic loci of the SRAP molecular marker in 780 Pinus yunnanensis Franch samples from the whole distribution area were used to establish a high-quality P. yunnanensis timber germplasm bank. Four P. yunnanensis germplasm subsets were constructed by improved least distance stepwise sampling at four sampling proportions, i.e., 10%, 20%, 30%, and 40%. Results showed that polymorphic locus retention rate of the four germplasm subsets was above 90% and the allele retention rate was greater than 95%. The minimum values of the five genetic diversity parameters were obtained from the germplasm subset constructed using the 10% sampling proportion, and the genetic diversity in the population of this subset was smaller than that of the original germplasm. Mean t-tests between the original and germplasm subsets showed significant differences between the 10% sampling ratio germplasm subset and the original. Furthermore, for the variance t-tests, several indicators of genetic diversity demonstrated significant differences between the original germplasm and the 10% and 20% germplasm subsets, respectively, and the variances of these germplasm subsets were smaller than those of the original. The correlation coefficients of the mean genetic diversity between the original germplasm and the four germplasm subsets all reached 0.99. The correlation coefficients of genetic diversity between the original and the 40% and 30% germplasm subsets were more than 0.80. The average genetic distance of the 30% germplasm subset was 52.16% higher than that of the original germplasm; at the same time, the distribution pattern of variance components of the original germplasm was well maintained and similar clustering to the original germplasm was achieved with a large genetic distance. Therefore, the 30% sampling proportion germplasm subset could be used as a representative subset of the germplasm resources of P. yunnanensis.