In Morocco, walnut tree (Juglans regia L.) cultivation is ancestral with complicated misidentification and unknown local genetic diversity. Atlas Mountains host a rich walnut tree germplasm, which has arisen from seedlings. The use of molecular markers makes it possible to overcomes confusion usually encountered in phenotypic characterization, thereby elucidating the real genetic diversity magnitude. This study aimed to investigate the genetic relationships among 33 local walnut trees grown in two contrasted agroecosystems, while comparing them with 8 Bulgarian varieties belonging to an ex-situ collection, using ISSR markers. The use of 13 primers generated 120 reproducible ISSR markers, with 7 to 13 bands and an average of nine bands per primer. This number reflected the high level of polymorphism between genotypes revealed by the selected primers. The average polymorphism rate was 75.2%, with the polymorphic information content varying from 0.212 to 0.370 and with an average of 0.32. Genetic distances and the pairwise comparison showed that all pairs of genotypes are distinct using more than 10 markers with a maximum of 50 bands. The dendrogram showed an obvious phylo-geographic structuring and the differentiation of genotypes by origin area. The Bulgarian cultivars form distinct groups that confirms the specificity of the Moroccan walnut gene pool. However, the dendrogram obtained showed that the Moroccan genotypes are genetically close to a certain number of Bulgarian genotypes. This indicates that some Moroccan genotypes and the Bulgarian varieties could have a common ancestor.