Salix gordejevii (Salicaceae) is a climax and dominant sand-fixing shrub species native to the northern China. We assessed S. gordejevii population genetic variation in different environmental gradients in Horqin Sandy Land, Northern China using inter-simple sequence repeat (ISSR) markers, and investigated the possible existence of relationships between genetic diversity and environmental gradients. The results showed that S. gordejevii populations in general have high genetic diversity. An analysis of molecular variation (AMOVA) revealed relatively high levels (> 89.91%) of within-population genetic variation. Based on cluster analysis, the 12 studied S. gordejevii populations can be clustered into three clades. Genetic diversity and differentiation of S. gordejevii populations are affected from different environmental gradients. Genetic diversity of all populations was affected by habitat environment change, and was well-correlated with the humidity gradients. These results have important implications for restoration and management of degraded ecosystems in arid and semi-arid areas.