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Evaluating genetic diversity in walnut (Juglans regia L.) populations is a rapid approach used by walnut breeding programs to distinguish superior genotypes. The present study was conducted on the walnut population of Hamedan Province, one of the richest and most genetically diverse regions in Iran, during 2018–2019. After the initial screening, 47 genotypes were selected for further evaluation of pomological and phenological traits based on International Plant Genetic Resources Institute (IPGRI) descriptors. Nut and kernel weights among the selected genotypes ranged from 7.15 to 21.05 g and 3.0 to 10.8 g, respectively. Principal component analysis (PCA) categorized the genotypes into three distinct groups. Whereas the cluster analysis (CA) revealed the similarities and dissimilarities among the genotypes by identifying four major clusters. Spearman correlation analysis showed a positive correlation (p < 0.01) between nut weight (NWT), nut size, and kernel weight (KW), while a negative correlation (p < 0.01) between shell thickness (STH) and packing tissue thickness (PTT) with kernel percentage (KP) was observed. Lastly, 10 of 47 genotypes (TAL8, TAL9, TAL10, TAL14, TAL19, TAL22, TB2, TB4, TB6, and RDGH5) were considered superior. Superior genotypes were late-leafing (25–40 days after the standard) and displayed a lateral bearing (LB) habit with heavy nuts (12.52–16.82 g) and kernels (6.53–8.15 g), thin shells (1.06–1.25 mm), and lightly colored kernels.

The development of late-leafing genotypes is the greatest strategy to deal with the late-spring frosts (LSFs). Due to the long breeding cycle, marker-assisted selection (MAS) is a fast strategy for screening walnut populations in breeding programs. The first aim of this study was to validate Turkish molecular markers associated with leafing date on Iranian walnut population. In addition, our final goal was to implement MAS on the Iranian population and identify the late-leafing genotypes. For this purpose, validation of two polymorphic SSR primers, including JRHR209732 and CUJ-RBO12, was determined by 14 early- and late-leafing cultivars/genotypes. Then, MAS was implemented for a population of 91 Iranian walnuts obtained from a breeding program. The results showed that the markers introduced on the walnut population of Turkey were able to distinguish the walnut population originated from Iran. This shows that the walnut populations of these two countries are genetically close to each other. In addition, the results showed that alleles with band sizes of 277 and 115 were related to late-leafing, and band sizes of 289 and 92 to early-leafing. The phenological data of three consecutive years of leafing date and validation and implementation of MAS were consistent according to SSR analysis. SSR analysis identified 14 alleles for all accessions, with allele numbers ranging from 5 (CUJRBO12) to 9 (JRHR209732) and an av-erage value of 7 alleles per locus. All analyses showed a high level of leafing time variation in our studied population, and cluster analysis (CA) revealed the similarities and dissimilarities among the accessions by identifying four major clusters. Finally, based on screening by late-leaf alleles, geno-types Ch35T12, ChLa1, ChFr2, ChFr3, Ped35T1, Ped35T4, PedLa1, PedLa3, PedOp1, and ChPed2 were introduced as late-leafing genotypes.