Genetic diversity of Stipa breviflora populations in the Inner Mongolian grasslands of China and its possible correlation with climatic factors was examined using geographic information systems and random amplified polymorphism DNA analysis. A total of 308 bands were produced with 28 arbitrary decamer oligonucleotide. Three major findings were demonstrated. First, the genetic diversity of S. breviflora was high but lower than that of Stipa grandis and Stipa krylovii. Second, genetic distances between the populations analyzed using the unweighted pair group method and the Mantel test had a highly positive correlation with geographical distances, indicating that spatial separation of this species in the studied area produced genetic shift in the population. Finally, both canonical correspondence and Pearson’s analyses revealed strong correlations between genetic differentiation and temperature in the area. We therefore conclude that temperature variations play an important role in genetic differentiations among the investigated S. breviflora populations.