Investigating the genetic mechanisms of local adaptation is critical to understanding how species adapt to heterogeneous environments. In the present study, we analyzed restriction site-associated DNA sequencing (RADseq) data in order to explore genetic diversity, genetic structure, genetic differentiation, and local adaptation ofStipa breviflora. In total, 135 individual plants were sequenced and 25,786 polymorphic loci were obtained. We found low genetic diversity (He= 0.1284) within populations ofS. breviflora. Four genetic clusters were identified along its distribution range. The Mantel test, partial Mantel test, and multiple matrix regression with randomization (MMRR) indicate that population differentiation was caused by both geographic distance and environmental factors. Through theFSToutlier test and environmental association analysis (EAA), 113 candidate loci were identified as putatively adaptive loci.RPK2and CPRF1, which are associated with meristem maintenance and light responsiveness, respectively, were annotated. To explore the effects of climatic factors on genetic differentiation and local adaptation ofS. breviflora, gradient forest (GF) analysis was applied to 25,786 single nucleotide polymorphisms (SNPs) and 113 candidate loci, respectively. The results showed that both temperature and precipitation affected the genetic differentiation ofS. breviflora, and precipitation was strongly related to local adaptation. Our study provides a theoretical basis for understanding the local adaptation ofS. breviflora.