Objective: The genus Datura (Solanaceae), comprising two species in Iran, includes toxic yet medicinal plants found in temperate and tropical regions. Despite their wide distribution, morphological and anatomical studies of these species remain scarce, with most research focused on phytochemical composition. This study aims to comprehensively evaluate the morphological and anatomical features of leaves and stems of D. stramonium and D. innoxia to identify reliable diagnostic characters for species discrimination.

Method: Quantitative and qualitative traits of vegetative and reproductive organs were assessed for 3–6 individuals from each of 37 populations across Iran. Leaf and stem cross-sections were manually prepared and stained with Toluidine blue O. Multivariate statistical methods, including PCA, UPGMA clustering, and PCoA, were employed to evaluate the observed variation.

Results: D. innoxia and D. stramonium differed significantly in several morphological traits. Leaf margin shape, leaf type, fruit type, hair presence/absence, petiole length, leaf dimensions, and fruit dimensions showed diagnostic value. Anatomically, midrib dimensions, phloem and xylem thickness, palisade and spongy parenchyma thickness, and adaxial epidermal thickness were important leaf characters. Stem anatomical features—including phloem thickness, parenchyma thickness, trichome size and presence/absence, collenchyma thickness, and xylem thickness—also provided valuable diagnostic criteria. Notably, Iranian D. stramonium populations were consistently glabrous, contrasting with previous reports from other regions.

Conclusions: The two species formed distinct, well-separated clusters in all multivariate analyses. Inter- and intra-specific variations, including population-level differentiation, are discussed. The diagnostic characters identified provide a robust framework for species identification and contribute to the systematic understanding of Datura in Southwest Asia