Safflower ( Carthamus tinctorius ) is an oilseed crop which is largely affected by the damage of safflower fly ( Acanthiophilus helianthi ). Some of the physical traits related to the seed, including the color and hardness of the seed coat, have been introduced as factors affecting the mechanisms of resistance to pests ( Antibiosis and Antixenosis ) as well as the agronomic traits and nutritional and medicinal value of different plant species. Since the seed coat in different species of the genus Carthamus has several color patterns, therefore, after collecting a germplasm consisting of cultivated species (with white seed coat), four wild species (white, brown and black colors) and a breeding line obtained via interspecific hybridization with black seed coat (A82), the effect of color, hardness, surface structure, thickness and polyphenolic composition of the seed coat on the resistance to safflower fly was investigated. Also, the effect of seed coat color on the antioxidant properties, total flavonoid content (TFC), cyanidin-3-glucoside content (Cyd-3-glu), quantity and quality of seed oil was investigated. Then, the possibility of transferring seed color through intra-specific crosses was studied and inheritance of seed coat color were considered. Subsequently, based on three candidate genes in the anthocyanin biosynthesis pathway, the molecular basis of seed coat color was studied. Also, the effect of consumption of black-coated seed genotype, A82, compared to white-coated seeds from cultivated genotypes was investigated on biochemical enzymes of blood serum and vital tissues of Wistar rats. The choice test in 2014 revealed the significantly higher values of the percentage of infested heads, seed loss and seed yield loss in cultivated safflower compared to wild accessions and the line A82. Similar results were obtained in independent choice tests in the following two consecutive years, and the percentage of seed loss and seed yield loss in the black-seeded genotype of A82 was significantly lower than the other four cultivated genotypes. Additionally, the highly significant correlation was observed between the resistance to safflower fly and each of the two parameters of seed hardness and seed coat color. Therefore, the cultivated genotypes with white seed coats were more sensitive to damage of safflower flies and showed the least resistance compared to wild genotypes with colored seed coat and genotype A82 due to lower thickness of seed coat. Based on the differences observed in the seed coat structure, surface and thickness, it is suggested that the morphological structure, hardness and seeds coat color concomitantly contribute to the fly resistance through the antixenosis mechanism. Seed coat phytochemical studies showed that TFC and Cyd-3-glu content probably did not interfere with seed hardness and antixenosis mechanism. However a significantly negative and positive correlations were observed between the concentrations of four polyphenolic compounds (rutin, apigenin, quercetin and ferulic acid) and Cyd-3-glu content with the resistance to safflower flies, respectively. In fact, cultivars with the highest concentrations of polyphenol compounds and the absence of anthocyanin in the seed coat showed the most damage to the safflower fly compared to genotypes with colored seed coat. Also, phytochemical evaluation showed that genotypes with black seed coat had high antioxidant properties. Based on the results of phytochemical tests, it seems that the inter-specific crosses in safflower followed by the creation of a new pattern in the seed coat color, did not have a negative effect on the oil content and the fatty acids composition. After intra-specific crosses and production of hybrid and progenies of F 2 , the results showed that genotype A82, as a pollinator was more successful in transferring seed color and probably two genes with an epistatic effect control seed color trait. Molecular analysis of seed color on the basis of nucleotide and peptide sequences showed that all three pal , chs and ufgt genes were found in genotypes with white and black seed coat, but probably, with the exception of the first two genes, ufgt gene is not expressed in white-seeded genotypes. The results of the HPLC analysis and the content of Cyd-3-glu confirm this result. The results of the study of the effect of consumption of black-coated seed genotype of A82 in comparison with the white-seeded cultivated genotypes (C111 and AC-Stirling) showed that, the consumption of safflower seeds, especially genotype A82, have not any negative and toxic effects on vital tissues of the liver, kidney and testis of Wistar rats.