One of the top-selling medicinal products worldwide is Hypericum perforatum (St. John's Wort). Despite its cosmopolitan distribution and utilization, little is known regarding the relationship of the bioactive compounds in H. perforatum to the plants from which they are purportedly derived. In this study, amplified fragment length polymorphism (AFLP) analysis of 56 Hypericum accessions, representing 11 species, was conducted to gain a better understanding of diversity within Hypericum species, especially within cultivated accessions of H. perforatum, and to establish a molecular methodology that will provide breeders and regulators with a simple, affordable, and accurate tool with which to identify purported H. perforatummaterial. Utilizing four primer combinations, a total of 298 polymorphic markers were generated, of which 17 were present in all H. perforatum accessions and 2 were specific to only H. perforatum. This study demonstrates that AFLP can be utilized not only to determine the relationships of closely related Hypericumaccessions, but as a tool to authenticate material in herbal remedies through the use of genetic fingerprinting.