Olive (Olea europaea L.) is a key agricultural species in Mediterranean and semi-arid regions, where optimizing fruit and oil traits is crucial for both economic and nutritional value. This study investigates the impact of cultivar genetics and location on fruit characteristics, oil yield, quality parameters, and fatty acid composition in four olive cultivars-Picual, Sebhawy, Chemlali, and Manzanillo-cultivated in El-Sheikh Zuweid and El-Qantara. ElSheikh Zuweid favored larger fruit sizes, higher oil content (21.58 +/- 1.52%), and monounsaturated fatty acids (MUFA, 70.49 +/- 1.56%), while El-Qantara exhibited higher polyunsaturated fatty acids (PUFA, 13.74 +/- 2.79%) and linoleic acid (12.22 +/- 2.80%). Among cultivars, 'Picual' excelled in fruit morphology, while 'Sebhawy' achieved the highest oil content (22.52 +/- 1.29%), total soluble solids (23.05 +/- 0.73%), and polyphenols (0.068 +/- 0.010 g 100 g-1). 'Chemlali' recorded the highest MUFA (69.57 +/- 1.34%) and oleic acid (67.58 +/- 2.87%), while 'Sebhawy' had the highest PUFA (14.64 +/- 1.80%). ANOVA confirmed significant effects of location and cultivar on most traits, with interaction effects observed for total soluble solids and fatty acid composition. Genetic analyses using inter simple sequence repeat (ISSR) and start codon targeted (SCoT) markers showed moderate to high polymorphism, identifying 114 and 178 loci, respectively, with a combined Shannon diversity index of 7.33. Principal component analysis (PCA) and k-means clustering highlighted associations between specific molecular bands and desirable traits, such as fruit weight and oleic acid content. With 'Sebhawy' excelling in oil-related traits and 'Picual' demonstrating superior morphological adaptability in favorable conditions, these findings emphasize the role of genetic and environmental factors in olive trait development and the value of integrating morphological and molecular data for breeding programs.