Walnut is one of the most important nuts regarding their production and consumption. The available but uncharacterized genetic resources of walnut are important for the development and breeding of local varieties. Greece holds an important number of genetically uncharacterized walnut landraces, especially within the area of Parnon, which is considered to play a significant role as an in situ gene bank, due to its unique location traits. However, the genetic characterization and further use of these resources has been insufficient, due to the absence of genetic studies. In this study, we implemented SSR molecular markers, both to genetically characterize the walnut tree genetic diversity of the Parnon area and to identify its unique genetic structure, which will form the starting material for subsequent breeding programs. Overall, high levels of genetic variation were found among the individual walnut accessions that were collected in the Parnon mountain region.