Plant genetic diversity is the fundamental of plant-breeding programs to improve desirable characteristics. Hence, evaluation of genetic diversity is the first step in fruit-breeding programs. Accordingly, the current study was carried out to evaluate 25 superior walnut genotypes in respect of phenotypic and cytological characteristics. For this purpose, 560 walnut genotypes in southwest of Iran were evaluated based on UPOV and International Plant Genetic Resources Institute (IPGRI) descriptor. After a 2-year primary evaluation, 25 superior genotypes were selected for future phenotypic and genome size assessment. Flow cytometry was used to estimate genome size of the selected superior genotypes. A high genetic diversity was found in walnut population collected from the southwest of Iran. The selected superior genotypes had high yield, lateral bearing, thin-shell thickness (0.90–1.64 mm), high nut (12.54–19.80 g) and kernel (7.02–9.91 g) weight with light (L) to extra light (EL) kernel color which easily can be removed from the shell. Also, FaBaCh2 genotype turned out to be protogynous being important as a pollinizer cultivar. In addition to extensive phenotypic analysis, genome size was determined. The studied genotypes were diploid (2n = 2x = 32) and varied in genome size from 1.29 (FaBaAv2) to 1.40 pg (FaBaNs12). Correlation analysis showed that lateral bearing, budbreak date, nut size, and weight were the main variables contributing to walnut production. A linear relationship was found between genome size and nut weight (r = 0.527**), kernel weight (r = 0.551**), and nut size index (NSI) (r = 0.487**). Therefore, genome size can be considered as a strong and valuable tool to predict nut and kernel weight and nut size.