The rising interest in medicinal plants has brought several species of the genus Echinacea to the attention of many scientists. Echinacea angustifolia, E. pallida, and E. purpurea are the most important for their immunological properties, well known and widely used by the native Americans. The three species are easily distinguishable on the basis of their morphological characteristics, but it would be difficult, if not impossible, to distinguish them in commercial preparations of ground, dry plant parts of E. purpurea (the most valuable species for chemotherapeutic properties) mixed with the other two species. Species-specific molecular markers could be useful to address this issue. In the present work, using fresh material collected from cultivated Echinacea spp., AFLP analysis was used to discriminate the three species and to detect species-specific DNA fragments. By using 14 primer combinations it was possible to detect a total of 994 fragments, of which 565 were polymorphic. Overall, 89 fragments were unique to E. purpurea, 32 to E. angustifolia, and 26 to E. pallida. E+CAC/M+AAT or E+CAC/M+AGC alone provided 13, 9, and 4 or 7, 5, and 5 specific fragments for E. purpurea, E. angustifolia, and E. pallida, respectively. A validation trial to confirm the results was carried out on bulked samples of 23 accessions covering most of the genetic diversity of the three species. The results are discussed in terms of practical applications in the field of popular medicine, detecting frauds, and implications for the genus Echinacea.