Black henbane (Hyoscyamus niger L.) is frequently cultivated as a rich source of tropane alkaloids particularly hyoscyamine and hyoscine (Scopolamine) which are extracted from its whole aerial herb including dried leaves and flowering tops. These valuable natural compounds are widely used in modern medicinal preparations as well as pharmaceutical industries in India and around the world. In the present study, genetic divergence among twenty-nine accessions of Hyoscyamus niger L. was evaluated for the nature along with amount of genetic diversity for eleven economic traits. Accordingly, all the twenty-nine accessions were grouped into eight diverse clusters (cluster I-VIII). The enormous diversity was also indicated by the wide range of D²-values (4.032 to 544.535). A number of unique accessions HN-30, HN-31, HN-15, HN-22-Y, and HN-29-Y were identified that may be interesting genotypes for the genetic improvement of the different morphometric and tropane alkaloid traits. Therefore, these accessions can be further exploited in future hybridization programs for the development of a new variety/cultivar for commercial productions. Manuscript profile