In the present study, HPLC technique was applied to determine scopolamine and hyoscyamine contents in five Hyoscyamus species including H.niger L., H.reticulatus L., H.pusillus L., H.arachnoideus Pojark., and H.kurdicus Bornm., collected from different geographical origins of North West of Iran. The range of genetic similarity was obtained between 91.07 and 99.89 within Hyoscyamus accessions based on scopolamine and hyoscyamine alkaloids composition. Estimated heritability was high for both traits and ranged from 0.82 to 0.99. The existance of high GCV indicates the genetical control of tropane alkaloids which leads us to achivement of genetic improvement by selection of desirable plants for breeding. Our data also implies the positive assosiation between N, P, K, Ca & EC and alkaloids yield in plants which will be useful for production programs in the future.