Abstract Background Angelica dahurica (Apiaceae) is an important herb in traditional Chinese medicine. Because of its important medicinal and economic values, its wild resources were over-exploited and increasingly reduced. Meanwhile, the diversity of cultivars of A. dahurica has decreased as a result of long-term artificial cultivation. However, there are no population genetics studies of natural A. dahurica reported yet, especially for using microsatellite markers (SSRs) to investigate population genetics of the species. Results Sixteen polymorphic EST-SSR loci were isolated from A. dahurica with transcriptome sequencing technology (RNA-Seq). The number of alleles varied from 2 to 15 per polymorphic locus over populations with the observed and expected heterozygosities ranging from 0.000 to 1.000 and from 0.000 to 0.829, respectively. Significant deviations from Hardy–Weinberg equilibrium were observed at 8 loci. Tests of linkage disequilibrium showed 11 informative locus pairs were significant across all populations. Cross-species amplification showed that 14 out of 16 SSR loci have the transferability in cultivar-A. dahurica cv. ‘Hangbaizhi’ and A. decursiva. Conclusions The 16 newly developed loci microsatellite primers with RNA-Seq will be useful for further investigating population genetics of A. dahurica, cultivars and other members of this genus.