The study was carried out to determine phenolic compounds, antioxidant activity, total lipids, saturated fatty acids, unsaturated fatty acids, and volatile organic compounds (VOCs) of 18 different walnut (Juglans regia L.) cultivars and genotypes grown in Usak province, Turkey. The biochemical compounds were identified for each cultivar and genotype and a comparative evaluation was carried out. The results showed that most of the biochemical compounds are significantly varying among each other (p < 0.05). The existence and abundance of the biochemical compounds in the cultivars and genotypes have not only found to be a cause of differentiation, but they have been important parameters for the similarities among the cultivars and genotypes. Total phenolic content and antioxidant activity ranged from 750.67 to 1245.64 mg GAE ml−1 and from 42.46 to 56.50% in kernels, respectively. The highest contents of the phenolic compounds were noted from gallic acid (11991 mg kg−1), ellagic acid (1057.16 mg kg−1), catechin (425.4 mg kg−1), and rutin (216.6 mg kg−1), as an average of all cultivars and genotypes. The highest lipid contents were noted from ‘Franquette’ and ‘Fernette’ as 64.28% and 63.26%, respectively, while the lowest content was noted from ‘Oguzlar 77’ (52.52%). A total of 42 VOCs were described from the 18 different walnut cultivars and genotypes. Overall, results showed that the phenolic profiles, fatty acids composition, and VOCs play an important role in similarities and diversities among the cultivars. According to the results, 7 different clusters were developed from the 18 cultivars and genotypes. Herein, ‘Fernor’, ‘Maras 18’, ‘Arslan Local Type’ and ‘Cisco’ cultivars were found to have the lowest biochemical compositions. The superior cultivars or genotypes, in terms of the abundance of the biochemical composition, were found to be ‘Gulizar Hanim Type’, ‘Lara’, ‘Pedro’, ‘Tulane’, ‘Fernette’, ‘Balkan’ and ‘Franquetta’, where they were found to develop 3 clusters by 1:3:3, respectively.