A cDNA encoding hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), a bifunctional enzyme catalyzing the last two steps in the scopolamine biosynthetic pathway, was isolated from Hyoscyamus senecionis, a medicinal plant endemic to the Iranian plateau. Expression analysis indicates that Hsh6h is expressed in all tested organs of H. senecionis including roots, rhizomes, leaves, stems and flowers unlike the other tropane alkaloid producing species. In parallel to this, in leaves, levels of scopolamine, the product of H6H, were higher than the substrate hyoscyamine. These data suggest that not only does the conversion of hyoscyamine to scopolamine take place in the root, followed by translocation to aerial parts, but also accumulated hyoscyamine in the aerial parts may be converted to scopolamine by activity of HsH6H. Analysis of expression profiles of putrescine N-methyltransferase and tropinone reductase I and II genes also indicates the organ-independent expression of these genes. Here we also introduce H. senecionis as an important tropane alkaloid producing species with its thick underground parts as a source of hyoscyamine, while its leaves can be considered as a source of scopolamine. Display omitted Production of tropane alkaloids and expression of tropane alkaloid pathway genes were presented in Hyoscyamus senecionis, for the first time. Organ-independent expression of newly identified h6h cDNA and the other TA pathway genes explain an atypical pattern of scopolamine accumulation in this species. •Cloning and characterization of a new H6H cDNA from Hyoscyamus senecionis.•An atypical pattern of tropane alkaloid accumulation and expression of TA pathway genes.•Hyoscyamine and scopolamine are mainly stored in rhizomes and leaves, respectively.