Descurainia sophia and Sisymbrium irio, both from Brassicaceae family have potential medicinal effects. Seeds are consumed widely across Iran in sweet drinks. To determine the morphological and molecular diversity, 55 genotypes from these two genera were studied. All 11 ISSR primers detected polymorphism and generated 274 polymorphic loci. The average values of Polymorphism Information Content (PIC), Marker Index (MI) and Resolving Power (Rp) for ISSR primers were 0.278, 6.899 and 11.57, respectively. The AMOVA analysis revealed a high genetic variation (58%) within each genus. The UPGMA clustering based on molecular analysis separated all 55 genotypes into two main groups, correlating with division of plants into two genera. Moreover,  seven morphological traits were evaluated   to distinguish all 55 genotypes. Among them, rosette figure showed the most significant differentiation between two genera. The highest correlation was observed between two traits of seed color and seed weight at 0.758. The dendrogram obtained from the morphological traits corresponded with the UPGMA clustering. The chemical composition of essential oils of D. sophia and S. irio were identified via gas chromatography mass spectrum (GC-MS). Although GC-MS analysis detected β-pinene as the dominant component in both plants but different compounds were also detected. The present investigation clearly indicates that these morphological traits alone or combined with molecular analysis using 11 ISSR markers is considered as the true reflection of two genera partitioning, and hence the eligibility of both molecular and morphological criterion are proved. These findings could be used for future breeding programs in fields of seed production and medicinal extracts.