This research was conducted to detect the morphological and molecular differences between golden henbane (Hyoscyamus aureus L) collected from south Syria. Seeds were collected from six locations, then sterilized and grown In Vitro. Grown plants were described for some morphological characters. The molecular characterization was carried out using the method of Inter Simple Sequence Repeats (ISSR) to detect genetic variations. The plants showed significant (p<0.01) morphological differences among studied locations, Plant height ranged from 14.97 to 18.97 cm and stem color ranged from red to green. Trichome density also ranged between medium, high and very high. Furthermore, significant differences in leaves dimensions were observed according to the plant geographical location. The main root length ranged from 6.09 to 8.37 cm. Molecular analysis by ISSR produced a total of 56 bands, 49 of which were polymorphic (87.5%), resulted from using 11 non-specific primers. A genetic distance dendrogram was drawn. based on the results of genetic characterization of plant from different locations. The plants collected from Salkhad separated with a genetic distance of 0.312 while the plants collected from other locations were grouped together, at the same time, they were divided into two sub groups, the first group comprised plants collected from Qalamoun and Dimas regions with a genetic distance of 0.032, whereas sub group II content Bosra plants which separated clearly from those of Dara' dam and Wadi Al-Zaidi by a genetic distance of 0.017, These results may indicate a relationship between genetic characters of this plant species with geographic distribution.