The genetic diversity of 50 seed watermelon germplasm from different places were analyzed by SSR molecular marker and the primary core collection was constructed by Least Distance Stepwise Sampling(LDSS).The results showed that:(1) 31 pairs of primers which showed high polymorphism were selected from 106 SSR primers, showed 115 polymorphic bands in 138 bands owning 83.33% polymorphic loci, the average of Shannon information index(I), Nei’s genetic diversity(h),effective number of alleles(N_e) and number of alleles(N_a) by POPGENE32 analysis was 0.419 3, 0.272 4, 1.458 0 and 1.981 4, respectively, which indicated high genetic diversity in 50 seed watermelon accessions. (2) The clustering result showed that genetic similarity coefficient ranged 0.57-0.91. Some germplasms distributed in different origins and far geographic distance showed high genetic similarity coefficient in 50 accessions. (3) The germplasm were sampled by the method of LDSS and ratio at 70%, 60%, 50%, 40%, 30%, 20% and 10%, and the genetic diversity parameters of each core subset was compared. The results showed that the change of genetic diversity parameters of each core subset was not clear with the samples reduced in six samplings. But the percentage of polymorphic loci, N_e, h and I of 10 primary accessions were obtained by sampling proportion 20%. It showed that the primary core collection from 20% sampling could well represent the genetic diversities of the whole collection．