Our study focused on the pollen morphology of 13 species of Matthiola and the closely related genus Dvorakia. We used light and scanning electron microscopes to examine the importance of pollen characteristics in classifying these genera. Pollen grains were acetolysed, measured, photographed, and described based on qualitative and quantitative characteristics. Here, we present statistical analyses and multivariate statistics for quantitative data. Pollen in both genera are tricolpate and varies in shape from subprolate to prolate. We distinguish two pollen types based on lumen width. Matthiola pollen has both reticulate and macroreticulate ornamentation, while Dvorakia alyssifolia pollen is reticulate. The thickness of the exine and the width of the mesocolpium are important traits in addition to exine ornamentation. Iranian Dvorakia and Matthiola species' pollen size can be divided into four types, one of which is Type I (Dvorakia alyssifolia type). Among the examined species, M. tomentosa has the smallest pollen grains, while D. alyssifolia has the largest grains. Pollen morphology confirms the transfer of D. alyssifolia from Matthiola to Dvorakia. Additionally, we propose a species key for the genera.