Abstarct: Abstract: Plant populations in breeding programs in their crops show segregation of traits from the F2 generation. Breeding techniques require sufficient information about the germination process and early seedling establishment. Therefore, in order to evaluate the initial vegetative growth and establishment of F2 generation seedlings of superior genotypes of barberry resulting from interspecific crosses, fruits of 12 selected genotypes from the F1 generation resulting from crosses of Berberis integerrima, B. cratagenia, and seedless commercial barberry were collected in the autumn season. In the first stage, seedling establishment and germination percentage were evaluated in three growing media including ½ peat moss + ½ perlite, ½ cocopeat + ½ perlite, and ⅓ peat moss + ⅓ cocopeat and ⅓ perlite, and after initial seedling growth, 11 morphological and growth traits were examined for diversity in 12 plant populations with codes 0903, 0308, 0525, 0601, 0539, 0534, 0528, 0530, 0516, 0532, 0038, 0607. The results of barberry germination showed a significant difference in populations in terms of germination power, with population 0607 achieving the highest germination rate (95%) and population 0903 having the lowest germination rate at 17%. Significant differences in germination rates of barberry seedlings were not observed in the growing media. The simple correlation coefficients between traits showed a positive and significant relationship between leaf number, plant height, leaf shape, leaf margin thorn size, leaf margin thorn number, first leaflet shape, and leaf margin arrangement. baxsed on PCA results (Principal Component Analysis), the first factor explains 88.8% of the variance while the first five factors explain 99.2% of the variance. In the first component, plant height trait primarily, and leaflet thorn size and thorn number had the highest eigenvalues in the second component. Genotype distribution in biplot analysis was determined in the first two components. Genotypes from populations 05-16, 06-07, 05-30 were placed in the first region, populations 05-28, 05-38 in the second region, populations 09-03, 05-39, 03-08 in the third region, and population 05-032 in the fourth region in the biplot analysis, among which populations 05-16, 05-28, and 05-39 were separated in the central groups. In cluster analysis, genotypes were divided into the first and second groups from a scale distance of 13. A total of 240 genotypes are in the first group and 248 genotypes in the second group. The findings indicate that barberry bushes in the F2 generation have considerable diversity in the initial growth stages, which can be utilized in breeding programs in the future.