Abstract: Safflower, Carthamus tinctorius, L. is an oilseed crop that belongs to the family Asteracea. The genus Carthamus is comprised of 25 species including the only cultivated species of Carthamus tinctorius. So far, the characterization of safflower using molecular markers has been limited. The objective of this study was to examine the cluster analysis of safflower accessions collected from different regions of Ethiopia using ISSR molecular markers.