Microsatellites (simple sequence repeats, SSRs) are co-dominant nuclear markers that are widely used in population genetic studies. Population genetic parameters from different studies might be significantly influenced by differences in marker number. In our study, 265 sequences with polymorphic microsatellites were obtained from SLAF-seq data. Then, subpopulations containing different numbers (5, 6, 7,…, 15, 20, 25, 30, 35, 40) of markers were genotyped 10 times to investigate the impact of marker numbers on population genetic diversity results. Our results show that genotyping with less than 11 or 12 microsatellite markers lead to significant deviations in the population genetic diversity or genetic structure results. In order to provide markers for population genetic and conservation studies for Rhododendron, 26 SSR primers were designed and validated in three species.