Cnidium officinale, Ligusticum chuanxiong and Angelica polymorpha have long been used as Chinese medicinal herbs to treat the same conditions. To elucidate the inter-specific relationships among these species, molecular and molecular cytogenetic analyses were conducted. Cytogenetically, C. officinale and A. polymorpha were found to be diploid with 2n=2x=22, but L. chuanxiong had a triploid chromosome number (2n= 3x=33). FISH analysis revealed that each two pairs of the 45S-and 5S rRNA genes were detected on the different chromosomes of C. officinale, and that triplet of the rRNA genes were localized on the same loci of the homologue in L. chuanxiong. GISH conducted using a genomic DNA probe detected strong cross-hybridization of genomes between C. officinale and L. chuanxiong, while no distinct GISH signal between C. officinale and A. polymorpha was observed. ITS and NTS data also revealed a very high sequence homology (95−96%) between C. officinale and L. chuanxiong. The RFLP profile of the rDNA-ITS enabled us to confirm the genetic distance among species. An effective species-specific SCAR marker for the identification of the wild plant, A. polymorpha, was also developed. Taken together, the results of this study suggest two possibilities; (1) L. chuanxiong originated from C. officinale, or (2) L. Chuanxiong and C. officinale diverged from the same parental plant.