The genetic diversity and genetic differentiation of eight Hippophae rhamnoides L. populations in eastern Gansu Province, as well as the effects of varying meteorologic conditions on them were assessed by using inter-simple sequence repeat (ISSR) molecular marker method. A total of 240 individuals in the populations were sampled. Using eleven primers, 165 bands were generated, ranging in size from 300 to 1500 bp, and 157 (95.76%) were found to be polymorphic. The analysis of molecular variance (AMOVA) demonstrated that there was a relatively high level (76.5%) of genetic variation within the populations, with the gene differentiation coefficient (Gst) and gene flow being 0.2418 and 1.5675, respectively. Therefore, to protect the gene resources of H. rhamnoides, the individuals within the populations should be first considered. Mantel test showed that genetic distance was significantly positively correlated with geographical distance (r = 0.65, p = 0.002), and regression modeling between genetic diversity and meteorologic factors suggested that there was a significant positive correlation between wind speed during blooming and genetic diversity of H. rhamnoides, illustrating that wind speed in blooming period and geographic distance were the vital factors affecting the genetic diversity of H. rhamnoides population.