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Safflower (Carthamus tinctorius L.) is mainly grown as oilseed crop in the arid and semi-arid regions of the world. The present study was aimed at estimating genetic variation of 48 genotypes belonging to three species (C. lanatus, C. oxyacanthus and C. tinctorius) using 109 EST-SSR primers pairs retrieved from two different sources. Forty two primer pairs produced polymorphic bands and yielded a total of 145 bands ranging from 100 bp to 500 bp with 2-8 alleles per locus. Polymorphic information content (PIC) values ranged from 0.04 to 0.695 with an average of 0.322. Cluster analysis clearly separated the species from one another. The cultivated safflower genotypes were divided into 4 subgroups each contained genotypes with almost same origin and genetic background. Results of analysis of molecular variance showed a significant difference between Iranian and exotic genotypes each of which possessing a highly intra-population variation. (c) 2012 Elsevier Ltd. All rights reserved.