One of the methods for breeding of fruit trees is the identification and selection of superior genotypes in different regions. Owing to climate changes in the recent years and the urgent need to changing the pattern of cultivation, it is essential to use adapted species to climatic conditions. Barberry is one of the plants that have been naturally grown in some areas of Isfahan province. For accessing the promising genotypes of barberry in Isfahan province, this research was carried out in the main areas of barberry growing. For this purpose, some physiological and pomological traits of about 100 barberry genotypes from different regions of Isfahan province were evaluated according to Union for the Protection of New Varieties of Plants descriptor. Evaluated traits were recorded in each year in four stages, including the physiological dormancy of the tree, the flowering, the vegetative growth of the tree as well as the ripening and harvesting time of berry stages from 2014 to 2016. According to the results, seven genotypes were selected, including Dolat Abad, Semirom, Dehaghan, Red leaf, and Evergreen genotypes as well as two wild genotypes. The results showed that the berry length ranged from 1.06 to 9 mm and the berry width ranged from 0.4 to 5.2 mm. The dried barberry per kilogram of fresh berries was between 200 and 700 g. The leaf length and leaf width of the studied genotypes were in the range 26–81 and 9–26 mm, respectively. The thorn length varied between 9 and 91 mm. The seed length and seed width were recorded in the range 0–4.5 and 0–2.6 mm, respectively. Dolat Abad and Dehaghan genotypes had the highest berry length and berry width, respectively. The most dried barberry per kilogram of fresh berries belonged to these two genotypes. Evergreen and Red leaf genotypes had the highest leaf length and leaf width, respectively. The highest seed length and seed width were observed in two wild genotypes of collected barberry. The results of correlation analysis of traits showed that tree growth vigor had a significant positive relationship with berry size, thorn length, seed length, and seed width. Berry firmness had negative correlation with berry length and berry width. On the basis of cluster analysis, genotypes were placed in three groups, so that genotypes from Dolat Abad, Semirom, and Dehaghan regions were placed in a same group. Red leaf and Evergreen genotypes with ornamental application were in another group, and two wild genotypes were also placed in the third group. Eventually, Dolat Abad, Semirom, and Dehaghan genotypes were introduced as the promising genotypes because of seedless berry with the highest berry length and berry width and the most dried barberry per kilogram of fresh berries.