A PCR-based DNA amplification method was applied to genetically distinguish the popular dietary supplement Hypericum perforatum L. (common St. John's Wort) from other related Hypericum species. Nuclear ribosomal gene sequences of the internal transcribed spacer (ITS) region were analyzed for 50 Hypericum taxa native to the Old and New Worlds, representing 11 of the 36 currently accepted taxonomic sections. This study provides a genetic method for authentication of commercial H. perforatum preparations. In addition, these data allow a preliminary assessment of phylogenetic relationships within the genus, revealing three strongly supported monophyletic clades, plus several secondary monophyletic groupings. Using ITS gene sequences, we were able to distinguish H. perforatum from all other species of Hypericum included in this study.