Background <p><i>Aconitum</i> is an important medicinal genus widely used in traditional Chinese medicine, which produces types of diterpenoid alkaloids (DA) among different species. We performed whole genome resequencing (WGS) research in <i>Aconitum</i> spp., and wish to find diterpenoid alkaloids related genetic variations.</p> Results <p>In this study, we re-sequenced 150 <i>Aconitum vilmorinianum (A. vilmorinianum)</i> including 102 from the cultivation garden and 48 from the wild, as well as nine wild samples of <i>Aconitum weixiense.</i> The intra-population differentiation of <i>A. vilmorinianum</i> was detected by evolutionary tree and population structure inference. We identify 47 DA biosynthesis genes that might be highly associated with the specialization of DA based on whole-genome resequencing. Of 616 significant SNPs and 105 significant InDels among these genes could be developed as polymorphic molecular markers capable of effectively recognizing <i>A. vilmorinianum</i> from <i>A. weixiense</i>. Furthermore, the significant SNPs and InDels were almost homozygous alternates in <i>A. weixiense</i>, whereas they tended to be homozygous references in the <i>A. vilmorinianum</i>.</p> Conclusions <p>Our results discussed the difference in genetic background in <i>A. vilmorinianum</i> compared to <i>A. weixiense</i> and these high-quality DA biosynthesis-associated polymorphic locus provided useful genetic information for discrimination of <i>A. vilmorinianum</i> and could serve as a vehicle to study the mechanism of DA differentiation in <i>Aconitum</i>.</p>