Dendrobium plants are one of the most valuable traditional Chinese medicinal herbs. Because of excessive exploitation and habitat degradation, Dendrobium species are endangered. Moreover, during no flowering stages, it is very difficult and even impossible to distinguish Dendrobium species based on morphological characteristics. Therefore, exploiting other DNA barcodes as complementary molecular marker for distinguishing Dendrobium spp. are necessary. Hence, developing an uncomplicated and exact method for authenticating Dendrobium spp. is imperative. In this study, nuclear ribosomal DNA internal transcribed spacer (ITS) sequences of 320 samples from 73 Dendrobium spp. were analyzed. The average intra-and inter-specific genetic divergences were 0.007 and 0.220, respectively. The TaxonGap method indicated that between-species variation of about 80.9% of the species were higher than within-species variation. The neighbor-joining tree showed that 73 Dendrobium spp. were divided into four major clades. Phylogenetic relationships between Dendrobium spp. supported traditional morphological methods and previously published molecular data. This study indicated that ITS region was not only suitable for species identification, but also for phylogenetic analysis of Dendrobium. However, it was also found that ITS could not readily resolve all species determination problems in Dendrobium.