Safflower, Carthamus tinctorius, L. is an oilseed crop that belongs to the family Asteraceae, arndiverse group of flowering plants that grow in many parts of the world. So far, therncharacterization of safflower using molecular markers has been limited. The objective of thisrnstudy was to investigate the genetic diversity of safflower accessions collected from differentrnregions of Ethiopia using ISSR molecular markers. For this purpose, seeds of seventy landracernaccessions collected from four administrative regions of Ethiopia (Amhara, Oromia, Tigray andrnSNNPR) were obtained from the IBC and grown in greenhouse at Addis Ababa University,rnFaculty of Life Science. DNA was extracted from a bulk leaf sample of five randomly selectedrnplants per accession using a triple CTAB extraction technique. Four primers were selected. Thernfour selected ISSR primers produced a total of 43 bands across the 70 safflower accession. Thernnumber of amplified fragments with ISSR primers ranged from 6 to 14 per primer with varied inrnsize of 100 to 1000 base pairs. Out of the 43 bands detected, 87.5% were polymorphic. In termsrnof region, Oromia showed the highest percentage of polymorphism (86.1%) with Tigray havingrnthe least polymorphism (20.9%). Safflower population from Oromia has 0.32 and 0.48 generndiversity and Shannon diversity index, respectively whereas Amhara population of safflower hasrnshown 0.27 and 0.39 gene diversity and Shannon diversity index, respectively. AMOVA alsornshowed that 98.9% of the variation is attributed to within population while 1.1% is amongrnpopulations variation. Both UPGMA and neighbor joining trees based on Jaccardâ€™s similarityrncoefficient showed weak grouping among individuals collected from the same regions. The resultsrnreveal the presence of higher genetic diversity that deserves conservation attention andrnsustainable use strategy to improve the productivity of safflower.rnKey words: - Carthamus tinctorius L, genetic diversity, ISSR molecular marker,rnsafflower, polymorphism, Ethiopia.