Safflower (Carthamus tinctorius L.) is recommended one of the prospective oilseed crops to grow in different stress conditions. Wild relatives of safflower are considered a good source of genes to improve cultivated safflower for important traits such as drought and salinity tolerance, and increased seed yield. This research was conducted in three studies. In the first study, C. tinctorius, C. palaestinus and C. oxyacanthus were crossed with each other and three interspecific population progenies including C. tinctorius ×C. palaestinus (TP), C. tinctorius × C. oxyacanthus (TO) and C. oxyacanthus × C. palaestinus (OP) were developed. Three selection methods including single plant selection (), modified selected bulk (MSB) and modified bulk (MB) were applied at F3 generation, using observable criteria reported to have high correlation with yield. Lines were derived from each selection method were evaluated in F4 generation in terms of agro-phenological traits under water stress and non-stress conditions. and MSB methods resulted in higher mean values for agronomic traits compared to MB under both moisture conditions and were equally efficient in selecting better genotypes. Higher mean values of agronomic traits for the best single plant (derived from method) and the best three selected plants (derived from MSB) compared to the other plants in each line were observed and indicated that capitulum diameter (CD), number of capitula per plant (NCP), number of branches per plant (NB) and plant height (PH) could be good criteria for identifying superior genotypes in safflower under both moisture environments. For , a high percentage of agreement was observed between visual selection and selection based on actual yield. In the second study, selection methods and stress tolerance index (STI) were jointly practiced in F5 generation for all segregating populations. Genetic gain was obtained under both water stress and non-stressed conditions for several traits including seed yield. Response to selection in different interspecific populations under water stress and non-stress conditions identified suitable criteria for improving seed yield. The results of response to selection in segregating populations for measured traits showed that OP population had the highest values of genetic gain compared with the other two populatio so that capitulum diameter, number of seeds per plant, seed weight, capitulum weight and plant height had the most influence on response to selection in this population. Genetic gain in TO population was obtained through capitulum diameter, number of seeds per plant, seed weight, capitulum weight, number of capitula per plant and plant height. For TP population, the genetic gain was not as high as it was for the other two populations. Visual selection based on capitulum diameter and plant height for OP and TO genotypes, and number of branches per plant and number of capitula per plant for TP genotypes would bring genetic gain in the next segregating generations, respectively. It is concluded that combining STI with selection methods in safflower interspecific breeding may efficiently identify superior genotypes in the early segregating generations in water stress and non-stress growing conditions. The third study was conducted to do phylogenetic analysis of Carthamus species base on fad2 gene sequences and also to determine the fatty acid composition of two segregating populations developed in study two plus 45 genotypes representing ten Carthamus species. Phylogenetic analysis suggested that the allotetraploid species C. lanatus had contributed in fad2-1 gene introgression into allohexaploid species C. creticus and C. turkestanicus. On the other hand, based on the complete sequence of fad2-1 gene, it was revealed that C. palaestinus was the closest relative species to C. tinctorius. The present study confirmed deletion of one base pair in fad2-1 gene encoding region in C. tinctorius genotypes led to the high oleic acid phenotypes. Semi-transgressive segregation was observed for fatty acid compositions in recombinant lines of the two segregating populations studied. Recombinant lines TP7, TP18, TP20, TP21 and TO7 derived from single plant selection method were identified as lines with high potential yield, high drought tolerance and high linoleic acid content. Key words: Carthamus genus, Wild relatives, Interspecific hybridization, Genetic gain, Fatty acid composition, Phylogenetic analysis, fad2 gene