Seedless barberry (Berberis vulgaris L. var. asperma) is one of the medicinal plants only cultivated in eastern parts of IranIn present study, for the first time we investigated similarity between B. integerrima (native to eastern Iran and having seedy fruit) and seedless barberry on the basis of ploidy level by karyotype preparation and Flow cytometric analysis. B. integerrima and seedless barberry were tetraploid (2n=4x=56). Then, in order to study the factors preventing seed formation, pollination studies including self-pollination, reciprocal cross-pollination and open-pollination was carried out and the effects of individual treatments on seedy and seedless fruits, fruit set rate and fertility of flower were investigated in populations of seedless barberry and B. integerrima. Analysis of Variance indicated that pollination treatment in seedless and wild barberry produced seedless fruits and decreasing fruit set to %80. Open pollination results in normal fruit set and produced seedless and seedy fruits in cultivated and wild barberry respectively. Emasculation without pollination reduced fruit set to %40. Reciprocal hybridization between seedless and wild barberry resulted to seedy fruits as %6 and %90 respectively. The pollen tube growth in each type of pollination experiments and the pollen germination were also investigated that the results will be presented. Previous studies and results of present study support our hypothesis asserting B. integerrima is probably one of the major ancestors of seedless barberry.