Ornamental flowering cherry trees (Prunus species) are popular landscape plants that are used in residential and commercial landscapes throughout most temperate regions of the world. Most of the flowering cherry trees planted in the United States represent relatively few species. The U.S. National Arboretum has an ongoing breeding program aimed at broadening this base by developing new cultivars of ornamental cherry with disease and pest resistance, tolerance to environmental stresses, and superior ornamental characteristics. Knowledge of the genetic relationships among species would be useful in breeding and germplasm conservation efforts. However, the taxonomy of flowering cherry species and cultivars is complicated by differences in ploidy levels and intercrossing among species. We have used simple sequence repeat (SSR) markers developed for other Prunus species to screen a diverse collection of over 200 ornamental cherry genotypes representing 70 taxa in order to determine the genetic relationships among species, cultivars, and accessions. Data were generated from 9–12 primer pairs using an automated DNA genetic analyzer (ABI3770), and subjected to UPGMA cluster analysis. Extremely high levels of polymorphism were exhibited among the materials studied, thus indicating that ornamental flowering cherry germplasm has substantial inherent genetic diversity. This information, combined with traditional morphological characteristics, will be useful in determining genetic relationships among accessions in our collection and for predicting crossability of taxa.