Sophora alopecuroides (Faboideae) is an endemic species, mainly distributed in northwest China. However, the limited molecular markers range for this species hinders breeding and genetic studies. A total of 20,324 simple sequence repeat (SSR) markers were identified from 118,197 assembled transcripts and 18 highly polymorphic SSR markers were used to explore the genetic diversity and population structure of S. alopecuroides from 23 different geographical populations. A relatively low genetic diversity was found in S. alopecuroides based on mean values of the number of effective alleles (Ne = 1.81), expected heterozygosity (He = 0.39) and observed heterozygosity (Ho = 0.55). The results of AMOVA indicated higher levels of variation within populations than between populations. Bayesian-based cluster analysis, principal coordinates analysis and Neighbor-Joining phylogeny analysis roughly divided all genotypes into four major groups with some admixtures. Meanwhile, geographic barriers would have restricted gene flow between the northern and southern regions (separated by Tianshan Mountains), wherein the two relatively ancestral and independent clusters of S. alopecuroides occur. History trade and migration along the Silk Road would together have promoted the spread of S. alopecuroides from the western to the eastern regions of the northwest plateau in China, resulting in the current genetic diversity and population structure. The transcriptomic SSR markers provide a valuable resource for understanding the genetic diversity and population structure of S. alopecuroides, and will assist effective conservation management.