Masson pine (Pinus massoniana Lamb.) is one of the most economically important and widely distributed forest species in China. Understanding the genetic diversity and population structure of masson pine germplasms has crucial significance to effective collection, conservation, and utilization of the resource. In the present study, we explored the characteristics of the genetic diversity (H) and genetic structure between three typical seed orchards in South China and revealed the degree of genetic differentiation between different economic superior traits by 24 EST-SSR molecular markers. All the loci were polymorphic and showed several alleles per locus with a mean of 6 alleles and 168 rare alleles in the population. The mean value of polymorphism information content (PIC) was 0.7011, which indicated that the markers were informative. There were moderate genetic differentiation and genetic diversities (H) among the masson pine subpopulations. Most of the genetic variation existed within the population, and only a small part existed among the populations. However, although the genetic differentiation among populations only accounted for a small part, significant differences in pairwise genetic differentiation in different subpopulations occurred. And the estimated value of the migration coefficient indicated a low gene flow in our populations. STRUCTURE analysis showed 3 clusters grouped similar to the Unweighted Pair Group Method with Arithmetic (UPGMA) clustering and principal coordinates analysis (PCoA). The clusters were neither strictly classified by geographical region nor classified according to the traits, especially for the FG-NN group had a relatively pure structure. These results provided comprehensive information about the collection and protection of these valuable masson pine germplasm resources in southern China.