To compare the characteristic of chloroplast matK gene sequences of different Herba Dendrobium species and to authenticate inspected species, the matK gene sequences of 12 species (including 22 materials) and outgroup were amplified, cloned, and sequenced. Genomic DNA of Dendrobium plants was extracted using modified cetyltrimethyl ammonium bromide (CTAB) method. The matK gene sequences were about 1 410 bp in length. The variable sites were 51 while the parsim-informative sites were 11. There were nucleotides insertions and deletions in some species, in addition to transitions and transversions, such as in D. denneanum and D.chrysotoxum. Interspecies and different populations (varieties) of Dendrobium could be distinguished on phylogeny tree. The average genetic distance was 0.008, and the maximal and minimal genetic distances between Dendrobium species were 0.014 and 0.003, respectively. There were 8-20 variable sites between Dendrobium species. The genetic distance between populations (varieties) was 0.001, and there were 1-5 variable sites. Moreover, the 4 inspected materials were successfully authenticated. The database of chloroplast matK gene sequences of 12 species of Herba Dendrobii and inspected species could be used for the molecular authentication between Dendrobium species and populations. The matK gene sequence could be used as molecular maker for authentication of Herba Dendrobium.