Chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental species in the world due to the great diversity of inflorescence form and color. There has been increasing demands for various types of chrysanthemums, such as cut flowers, potted plants and bedding plants. However, the genomic studies of this species have been not extensively conducted relative to other ornamental species due to high levels of polyploidy (2n = 4x =36 or 2n = 6x = 54) and heterozygosity as well as large genome size. In this work, we developed a molecular tool for cultivar identification using simple sequence repeats (SSRs) and investigated genetic diversity in 127 chrysanthemum cultivars. Of the 150 SSR primer pairs tested in this study, 62 primers were obtained from previous studies, while 88 primers were designed using the unigene sequences of C. nankingense and the Expressed Sequence Tag (EST) sequences of C. morifolium in the NCBI database. Thirty SSR primers were selected based on polymorphism and banding patterns in a subset of 8 cultivars and used to amplify the DNA of 127 chrysanthemum cultivars. The UPGMA dendrogram based on these 30 SSR markers showed that most of chrysanthemum cultivars were divided into five clusters. These results will benefit chrysanthemum research community to develop elite cultivars.