Seedless barberry (Berberis vulgaris L. var. asperma) is one of the few crops that is only cultivated in eastern parts of Iran. As a new crop there has not been any study to identify phylogenetic relationships of this plant with other related species existing in Iran. In this study, Amplification fragment length polymorphism (AFLP) markers based on four selected primer combinations (EcoRI/Tru1I) were used to evaluate genetic variation and phylogenetic relationship among wild and cultivated barberry populations belonging to north east and eastern Iran. Two other species of ornamental barberry and one species of Mahonia aquifolium were also taken in this study. An Unweighted pair group method with arithmetic averages (UPGMA) dendrogram based on genetic distances clearly clustered each species, confirming phylogenetic relationships at the molecular level. These results can clarify the ambiguity in the relationship between Mahonia and Berberis genera. The heterozygosity index, principle coordinates analysis (PCoA), Fst Index and analysis of molecular variance (AMOVA) revealed a significant difference among wild barberry populations. As expected, observed variation within the cultivated barberry population was very low and close to zero. Moreover, morphological markers were used to evaluate variation and phylogenetic relationships among Berberis populations compared to results from AFLP markers by means of the Mantel correspondence test. No significant value was found by the mantel test between AFLP data and morphological markers. The lack of correlation between AFLP and morphological markers suggests low efficiency of identification key of Flora Iranica for classification and phylogenetic consideration of the Berberis family. Further molecular and morphological investigations are necessary to improve understanding of the relationships within species and genera of the Berberis family

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