Juglans regia L. is an edible fruit tree cultivated worldwide for its fruits and wood and as an urban tree. Globally, there is growing concern for preserving the genetic diversity of trees with high economic and ecological value. This study investigates the genetic diversity of J. regia in urban landscapes and assesses the quality of its walnut kernels as a local food product. An inventory of 150 trees from five populations in public green spaces in Lugoj, Caransebeș and Jupa, as well as two semi-natural hilly ecosystems in the Banat Region, was conducted. Molecular analyses showed that Directed Amplification of Minisatellite-region DNA (DAMD) markers were more effective than Start Codon Targeted (SCoT) markers, with a higher average polymorphism of 56.26%, compared to 49.44%. DAMD07 achieved 100% polymorphism and DAMD05 showed a strong balance between P.I.C. (0.35) and polymorphism (54.54%). Chemical analysis revealed the following contents in walnut kernels: protein (12.81% to 16.80%), lipids (60.39% to 69.08%), total polyphenols (5484.66 to 10,788.4 mg GAE/kg), copper (3.655 to 8.532 mg/kg), manganese (14.408 to 28.618 mg/kg), zinc (19.813 to 46.583 mg/kg), lead (1.204 to 2.27 mg/kg) and cadmium (0.03451 to 0.08065 mg/kg). These findings are critical for conservation efforts, urban forestry management and ensuring the quality and safety of walnut products derived from J. regia.