Sequences of the internal transcribed spacer (ITS) was compared in three seabuckthrons (Hippophae rhamnoides subsp. sinensis, H. tibetana and H. neurocarpa) distributed in Qinghai Province, then the systematic positions of 15 seabuckthron samples were analyzed with Elaeagnaceae angustifolia being outgroup. The results showed that ITS sequences in three seabuckthron species varied in length from 600 to 605 bp. ITS-1, 5.8S and ITS2 varied from 201 to 203 bp, 166 to 167 bp and 232 to 236 bp, respectively. The sequence divergence among three seabuckthorn species was also remarkably high. Cluster analysis based on ITS indicated that H. gonicocarpa subsp. litangensis and H. gonicocarpa subsp. goniocarpa were distinct and could be classified as H. gonicocarpa and H. litangensis. H. gyantsensis had the closest genetic relationship with H. salicifolia and the distant relationship with H. rhamnoides subsp. sinensis. The positions of nine subspecies of H. rhamnoides based on morphological classification were different from the results of ITS.