Chrysanthemum morifolium Ramat. is one of the major flowering crop plants worldwide. However, domestic chrysanthemum markets have recently faced a downturn. To stimulate related industries, breeding technologies and efficient protection systems using molecular markers must be established. However, high cost and intensive efforts are required to develop useful molecular markers for the chrysanthemum as it is a polyploid crop with highly complex genome organization. Thus, the aim of this research was to develop expressed sequence tag-based simple sequence repeat (EST-SSR) markers, which are applicable to the chrysanthemum breeding program and cultivar protection, based on next-generation sequencing technology. From the RNAseq data of the standard chrysanthemum cultivars ‘Jungwoon’ and ‘Seinoisei,’ we identified 31,121 SSR loci and further retrieved 1,846 polymorphic SSRs. To test the marker efficiency of the 1,846 SSRs, we first chose 50 of the SSRs and designed primers by using the flanking sequences. It is noted that the nine EST_SSR markers show a single band-like amplicon, which can be exploited in various genetic studies. We proceeded to polymorphism tests for those SSRs with 56 chrysanthemum cultivars, confirming that the average polymorphism index content (PIC) was 0.69±0.058. Among those, we found that six SSRs were sufficient to specify the genetic identities of 55 chrysanthemum cultivars, which may be useful for protections of the related cultivars, as well as breeding programs, in the future.