Numerical taxonomy and seed storage protein analysis of Hyoscyamus species of Iran was carried out with the aim to illustrate species inter-relationship and to check the sub-generic taxonomic treatment proposed for the genus. Cluster analysis of morphological and protein data grouped the species in three separate clusters which supports the relationships of H. niger with H. reticulatus and H. kurdicus with H. squarrosus and H. arachnoideus. Discriminant analysis produced three separate groups with 100% correctness of predicted versus actual membership of the species studied, supporting the sub-generic treatment of the genus Hyoscamus.