ABSTRACTSeedless barberry (Berberis vulgaris L. var. asperma) is cultivated in arid and semi-arid areas of Iran. It is widely used as a food additive. Thirty-nine morphological characteristics were evaluated to estimate the genetic variability among 42 barberry accessions during 2010–2012. Analysis of variance showed that all of the variables tested in examined accessions were significant (P ≤ 0.05), with a high level of variability. There were significant negative and positive correlations among some of the characteristics of the accessions. Ten major factors that accounted for 79% of variation were identified. Cluster analysis divided the accessions into five major groups based on all of variables analyzed. The three main groups were berry size, seedlessness, and vitamin C level. Using three main factors, the accessions were plotted in three dimensions; ‘R2N1’ had larger berries with a higher dry weight, while ‘R12N2’ had smaller leaves, a less dry weight, and more thorns. Quantitative and qualitative characteristics of the berries such as color, size, and shape, the number of seeds per 100 berries, and the number of berries per bunch, taste, and acidity showed a high level of variation among the studied accessions. The high genetic diversity among barberry accessions in our collection indicates that it is a valuable resource that can be used in barberry breeding programs in the future.