Based on rDNA ITS sequences of D. officinale and the other 37 species of Dendrobium, a pair of allele-specific diagnostic primers, TP-JB01S and TP-JB01X, were designed to authenticate D. officinale from the other species. Before the diagnostic PCR, the primer pair, P1 and P2, for amplifying the whole ITS region was used to validate template DNA and to obtain the appropriate template DNA for the diagnostic PCR. Diagnostic PCRs were performed using the diagnostic primers with the total DNAs of the original plants as a template. When the annealing temperature was raised to 66 degrees C, only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. The diagnostic PCRs have been repeated many times and have played an important role in authenticating the stems of D. officinale in China. Compared with the authentication method by sequencing DNA fragments, the allele-specific diagnostic PCR is not only simpler and time-saving but also practical and effective.