To identify Dendrobium species and to analyze the habitats of Dendrobium officinale， the suitable candidate DNA barcoding genes of Dendrobium were evaluated. In this study， the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA and chloroplast psbA-trnH gene were amplified and sequenced from sixty-six samples of D.chrysotoxum， D.nobile， D.primulinum and D.officinale. The D.officinale samples were collected from Hangzhou， Huoshan， Yunnan and Wenzhou. The intra-specific and inter-specific divergence of Dendrobium was calculated， and the identification efficiency was assessed using Barcoding Gap， NJ tree by K2P distance. The results showed the intra-specific divergence of ITS2 (0-0.005) were obviously lower than the inter-specific divergence (0.070-0.228)， and four species of Dendrobium were also accurately distinguished in NJ tree. Whereas， there is no Barcoding Gap on psbA-trnH， and it cannot distinguish D.nobile and D.primulinum in the NJ tree， and it can be used as a supplementary sequence in the identification of Dendrobium. ITS2 and psbA-trnH of D.officinale from four habitats have multiple mutation sites， which have the potential to identify the habitats of D.officinale.