Aconitum vilmorinianum is a very important Chinese traditional medicinal plant, which is one of the main raw materials in Yunnan Baiyao as an analgesic and anti-inflammatory agent. Recently, it has been threatened by overexploitation and human disturbances. Genetic background of this species is essential for the efficient conservation and rational utilization. Therefore, a total of 18 novel microsatellite markers were developed for A. vilmorinianum using next-generation sequencing technology. The number of alleles per locus ranged from 1 to 5, with a mean of 2.560, and the observed and expected heterozygosities ranged from 0 to 1.000 and from 0 to 0.733, respectively. These polymorphic microsatellite loci will be especially useful for genetics studies of this important medicinal plant.