Safflower, Carthamus tinctorius L. is a member of the family Compositae or Asteraceae. Nowadays, it is mainly cultivated for extract of edible oil. In recent years inter-specific hybridization between cultivated and wild species have been used to enhance genetic variation in plant populations and rendering genotypes resistant to biotic and abiotic stress environment. Breeding through inter-specific hybridization coupled with marker assisted selection could accelerate the breeding programs. Inter simple sequence repeats (ISSR) is a PCR- basedmarker in which 16-25 bp long microsatellites primers in a single primer PCR reaction amplify the inter- microsatellite regions. This technique has been extensively used in genetic diversity studies because they need no prior DNA sequence information and development costs are low. In this research, ISSR marker was employed to study genetic variation, determine parental genome contribution and segregation distortion of F 2 and F 6 populations derived from a cross between cultivated safflower, Carthamus tinctorius , genotype C 111 and an accession of wild species, C. oxyacanthus , genotype ISF II .The DNA from the two parents and individual F 2 and F 6 plants was extracted following CTAB method. Fifteen ISSR primers that showed polymorphism in parental genotypes were used in F 2 and F 6 populations whichproduced 58 polymorphic parental bands.Dice similarity indices were subjected to cluster analysis using Complete algorithm. The dendrogeram revealed two major groups. Group 1 included genotypes similar to C 111 and group 2 included genotypes similar to ISF II . The analysis of molecular variance (AMOVA) also showed that genetic variance between two parental groups within each populations was significant (P 0/001) and therefore genotypes could be discriminated based on similarity to parental genotypes.Therefore, in F 2 and F 6 populations, the number of progenies similar to C 111 was more than the number of progenies similar to ISF II . Out of 58 parental markers,the maximum, minimum and the average of bands were 40, 22 and 30, respectively in F 2 generation and in F 6 , they were 38,19 and 29.4 bands respectively. Results of this study showed that not all the parental markers were transmitted to F 2 and F 6 progenies. Also in both populations the average of inherited parental bands was higher from cultivated C 111 genotype than wild ISF II genotype. Chi-square test showed a segregation distortion in both F 2 an F 6 populations from 1:1 expected ratio at 0.01 probability level. Also inF 2 and F 6 populations, among 58 loci, 24 (41%) and 27 (47%) of loci were significantly deviated from the expected normal segregation ratio (P 0/01), respectively. In both populations, most of the distorted markers skewed toward the wild genotype. It seems that thetype of molecular marker, differences in genome size of parents, self-incompatibility and natural selection areprobably responsible for segregation distortion. The presence of genetic distortion in crosses of cultivated and wild safflower species may preclude the maximum variation to be expressed in breeding programs. Key words: Carthamustinctorius , C. oxyacanthus , Inter-specific hybridization, ISSR, Gentic diversity, Segregation distortion