The development of allelopathic crop cultivars for weed suppression has been increasingly recognised as one of the possible components in integrated weed management. Allelopathic plant interactions can have either a harmful or beneficial effect and are generally evaluated by testing some physiologic mechanisms that result in the inhibition/stimulation of seed germination, plant growth and development due to the presence of another plant. Allelochemicals can be present in several parts of plants including roots, rhizomes, leaves, stems, pollen, seeds and flowers. Allelochemicals are released into the environment by root exudation, leaching from aboveground parts, and volatilisation and/or by decomposition of plant material. A laboratory bioassay study was conducted to evaluated allelopathic interaction of wild and cultivated safflower seedlings using equal-compartment-agar method from september 2010 to 2011 in faculty of agriculture of Isfahan University of Technology, Iran. In this method 400 ml glass beakers were filled with 40 ml of 0.4 % water agar and then the agar surfaced divided into two equal compartments that were occupied separately with donor and receiver genotypes. This study has been designed at four parts for screening of allelopathy effect and response of nine cultivated genotypes of safflower follow as: Padide, Goldasht, Arak2811, Isfahan14, Koose, Saffire, Ac-sunset, AC-sterling cultivars and C 111 line and two wild safflower genotypes including Isfahan1 and cross 12. The first and the second experiments were done to find the best duration of exposure to exudates (including 3, 5, 7 10 days) and the best seedling densities (including groups of 4, 8, 12 16 seedlings) to maximize the allelopathy. The donor species at the first experiment was Isfahan1 against Padide cultivar as the allelochemical receiver species, but in the second one the donor and receiver species were Padide and Isfahan1, respectively. Results have shown maximum allelopathy activity at a density of 12 seedlings and a exposure of 5 days that was the same for the first and the second experiments although were stimulatory and inhibitory, respectively. The third study was evaluation of allelopathy potential of cultivated safflower on the wild genotypes but the forth one was to assay cultivated safflower sensitivity under wild genotypes allelochemicals. The results of the third study have proved C 111 and Isfahan14 cultivar had most inhibitory on Isfahan1 accession’s root length but motivator on shoot length. There was no inhibition on cross12 genotype, while C 111 line had the most stimulatory on cross12 genotype. In the forth experiment, the growth of Koose, Goldasht, Arak2811, AC-sunset and AC-sterling have been prevented under Isfahan1 allelochemical. Padide, Isfahan14, Saffire and C 111 had no significant response to Isfahan1 and cross12 genotypes allelochemicals, as a matter of fact these cultivars were resistance. The present study demonstrates that there is a considerable genetic variation of allelopathic activity in safflower germplasm. It is possible to breed for cultivars with enhanced allelopathic activity for weed suppression.