Denphal-type Dendrobium is the most popular orchid for cut flower and potted plant trade. To improve commercial traits, many novel cultivars have been produced through hybridization by commercial breeders. However, the genetic relationship of most cultivars is unclear, thus hindering the progress of Denphal-type Dendrobium breeding programs. Therefore, the development of molecular markers is encouraged to identify different cultivars. In this study, based on the transcriptome database of the Denphal-type Dendrobium ‘Red Bull’, the polymorphisms expressed sequence tag-derived simple sequence repeat (EST-SSR) were developed from 100 pairs EST-SSRs that were randomly selected from the EST-SSR database. The genetic relationship of 42 Denphal-type Dendrobium cultivars was analyzed according to the developed EST-SSRs. Then, the transferability of EST-SSRs was analyzed by performing a relationship analysis of 40 Dendrobium species. The results showed that a total of 5174 potential EST-SSR markers were identified with 4486 unigene sequences, and 5289 primer pairs were successfully designed. Of the selected 100 pairs of EST-SSRs, a total of 86 pairs produced the expected polymerase chain reaction products of the primary screening, 58 pairs produced the expected fragment size, and 20 pairs showed polymorphisms. Furthermore, the dendrogram of 42 cultivars showed that at a genetic distance of 0.15, the cultivars collected were grouped into five clusters of three major clusters and two minor clusters; all these clusters had the same characters of each cluster. The transferability analysis showed that 18 of the 20 EST-SSR markers among the 40 Dendrobium species were polymorphic. Overall, this study developed EST-SSR markers and will be valuable to facilitating genetic diversity in Denphal-type Dendrobium cultivars and Dendrobium species.