To understand the genetic relationship of Dendrobium in Qinba，the genetic diversity of 12 populations was analyzed using five intersimple sequence repeat（ISSR） markers．(1) The UPGMA analysis exhibited significant genetic differentiation among D.nobile Lindl，D.flexicaule Z. H. Tsi and D.hancockii Rolfe，which revealed that they are independent species．(2) Six populations of D. nobile were obviously divided into two branches，the populations of LJ, MT, YP and WF are classified as Clade I because of the close genetic consistency among them；the populations of YJ and PT are classified as CladeⅡdue to the highest degree of genetic consistency and genetic distance. (3) The Neis gene diversity (H_t) and coefficient of gene differentiation (G_st) of D. nobile were 0.388 and 0.934, respectively, which indicated that there were high diversity and gene differentiation among the populations of D. nobile．Our findings clearly evidenced the genetic diversity of D. nobile in different areas of Qinba, which will be used in identification and intellectual property protection．