In the current climate change scenarios, the choice of seed sources is one of the main factors affecting the establishment and productivity of forest tree plantations. Juglans regia L. (walnut) is one of the more valuable hardwood species since it could provide high quality timber and fruits. To obtain a commercially convenient production, different fruit varieties officially registered can be used; however these are not always suitable for any different climatic conditions. In the Italian country walnut is cultivated since ancient time and ecotypes adapted at various environmental condition still exist. We researched walnut provenances as putative biodiversity sources; this genetic material could be used to establish new seed orchards or hardwood plantations, as well as to recover the cultural tradition, productions and local economy of neglected rural areas. A multidisciplinary approach, integrating molecular markers (ISSR), morphological traits (equatorial and polar diameter, shape, dry weight) and biochemical composition of fruits(total oil, fatty acids, tocopherol) was applied to analyze several hundred samples collected in Central Italy sites (Campania, Abruzzo, Molise and Apulia regions). Particularly, samples of Abruzzo and Molise provenances were collected on centuries old walnut trees along over 200 Km of the Royal Tratturo Candela– Pescasseroli. Tratturi were the old grassy wide fixed routes, formed over centuries by transhumance, used for the seasonal migration of people and animals from the mountains to the coastal plains. In addition, samples of four registered varieties (two from Southern and two from Northern Italy) were also considered for comparison. Selected ISSR primers exhibited a strong ability to discriminate walnut provenances. The Principal Coordinate Analysis, performed on the ΦPT value, clustered the germplasm in distinct groups.The genotyping results were partially confirmed by the morphological and biochemical analysis of fruits. Two walnut provenances, one from a hilly plateau in Campania region, the other from mountainous zone of Abruzzo, shown different genetic, morphological and biochemical characters; they could be a promising source of seeds for nurseries and hardwood plantations.Cluster analysis (UPGMA dendrogram based on Nei's genetic distances) distinguished the northeasternprovenances, and clustered the 75% of the Candela–Pescasseroli provenances. The obtained results showed that the genetic differentiation of Italian walnut could be due to contrasting forces: domestication and selection, but also to intense inter-regional transfer of plant material by human activities like transhumance along the Tratturi. In the past, these paths had an enormous historical, economic, and cultural impact on social regional structure of Europe. In the second half of the twentieth century, the pastoral economy of these regions declined. In 2018 Italy, Austria and Greece proposed candidature of transhumance migratory routes as UNESCO Intangible Cultural Heritage.For Italy, the revaluation of the old grassy roads as Royal Tratturo is tight linked to the new interest for the rural development of internal areas located in the hilly and mountainous areas of Apennine. In this contest, our research on the "Walnut Tratturo", characterizes on the genetic resources, local products, traditions and the economy of rural communities, could be an important tool.