The genus Carduus L. comprised about 90 species, some of which are medicinally important and have high content of flavonoids, and phenolic acids. There are about nine Carduus species growing in Iran, out of which one species is endemic. Biosystematic investigation of the genus has been confined to morphological and palynological studies only and we do not have information on molecular study, species relationships and genetic structure of Carduus taxa in the country. We therefore, carried out multiple data set analyses of these species for the first time by using micro-morphological and molecular characteristics and provide data on species diversification and their affinities for the first time. The results showed that micro-morphological data can differentiate Carduus species, while the species relationship obtained by different approaches may disagree in some points. The results suggest that complexity observed in the taxonomy of the genus Carduus may be due to historical introgression events occurred between nuclear and chloroplast genomes during species diversification.