Stipa breviflora is a desert steppe plant species found widely in central Asia, and is a major forage plant in the desert steppe region of Inner Mongolia. The desert steppe is dominated by several Stipa species, including S. bungeana, S. klemenzii and S. krylovii. S. breviflora is adapted to a wide range of environment conditions, and tolerant to extremely tough climatic and soil conditions. The objective of the study was to estimate the genetic diversity of S. breviflora populations by studying different S. breviflora populations from 8 geographic locations in the Inner Mongolia steppe. Healthy and rustless leaves of S. breviflora individuals were sampled randomly at each site for RAPD analysis. Our results showed: (1) 28 primers were screened from 102 arbitrary primers with 10-bp. A total of 308 bands were produced, including 151 polymorphic bands, accounting for 49.03% of the total. There were 45 unique loci obtained, accounting for 14.61%. (2) The genetic diversity of S. breviflora (49.03%) was lower than that of S. grandis (54.75%) and S. krylovii (74.67%) reported earlier by other authors. (3) Eight different geographical populations could be grouped into 2 categories: warm-temperate and cold-temperate populations with 3 different methods, i.e. Hierarchical cluster analysis, PCA and UPGMA clustering. These 2 groups were mainly characterized by marked differences of thermal factors, corresponding to their spatial distribution boundaries. (4) Mantel test showed a significantly positive correlation between genetic distance and geographical distance (r=0.534，P＜0.05). (5) The percentage of unique loci of 8 populations showed no significant correlations with accumulative temperature above 10℃, aridity and annual mean precipitation, according to Pearson correlation analysis.