Dendrobium is one of the largest genera in Orchidaceae, comprising about 800-1500 species mainly distributed in tropical Asia, Australasia, and Australia. There are 74 species and two varieties of this genus in China. Because of their ornamental and commercial value, Dendrobium orchids have been studied at low taxonomic levels. However, structural changes and effective mutational hotspots of Dendrobium plastomes have rarely been documented. Here, 30 Dendrobium plastomes were compared, comprising 25 newly sequenced in this study and five previously published. Except for their differences in NDH genes, these plastomes shared identical gene content and order. Comparative analyses revealed that the variation in size of Dendroubium plastomes was associated with dramatically changed length of InDels. Furthermore, ten loci were identified as the top-ten mutational hotspots, whose sequence variability was almost unchanged with more than 10 plastomes sampled, suggesting that they may be powerful markers for Dendrobium species. In addition, primer pairs of 47 polymorphic microsatellites were developed. After assessing the mean BS values of all combinations derived from the top-ten hotspots, we recommend that the combination of five hotspots-trnT-trnL, rpl32-trnL, clpP-psbB, trnL intron, and rps16-trnQ-should be used in the phylogenetic and identification studies of Dendrobium.