In this study, the total DNA extraction method was modified based on the conventional CTAB method for Rhododendron, and then many microsatellite sequences were chosen from the published Rhododendron EST database and unpublished genome database of R.delavayi in our lab to design SSR primer pairs. Moreover, adding many SSR primer pairs referred to the references, 154 pairs of SSR primers were used to be screened. Finally, 26 pairs of polymorphic primers were screened out, and all of PCR amplicons were specific with clearer band and better repeatability. Furthermore, 10 pairs of polymorphic SSR primer fluorescentlylabeled were used to genetic diversity analysis on 69 Rhododendron germplasms. The results as following: mean effective allele number (N_e), polymophism information content (PIC), observed heterozygosity (H_O) , expective heterozygosity (H_E), and Neis gene diversity(H) for 10 SSR locus are 6.959 2, 0.795 2, 0.543 5, 0.826 5 and 0.820 2, respectively. Based on the consistency between the UPGMA clustering results and their pedigree analysis of Rhododendron hybrids, a speculation of ancestor parent types will be made for Rhododendron hybrids with unknown or unclear pedigree.