This experiment was conducted to assess genetic relationships among safflower genotypes from different geographical regions of Iran and other countries using newly developed simple sequence repeat (SSR) markers. By enrichment method, 32 primer pairs were designed of which 18 pairs were able to detect polymorphism in 105 safflower cultivars from Carthamus tinctorius, C. oxyacanthus, C. lanatus, C. glaucus, C. boissieri and C. dentatus. The selected SSR primers amplified a total of 59 alleles with an average of 3.27 alleles per locus among the cultivars and the average values of gene diversity, heterozygosity and PIC were 0.45, 0.37 and 0.39, respectively. Neighbour-joining cluster analysis based on Nei's genetic distance categorized populations of Carthamus in six major clusters; all wild accessions were grouped differently from cultivated genotypes. Cluster analysis significantly distinguished C. oxyacanthus genotypes in different categories: centre (Arak), northeast (Azarbaiejan), east (Kermanshah) and southeast (Shiraz, Chaharmahal and Kohgiluyeh). The presence of C. boissieri and C. glaucus in one cluster appeared to be in close relationship between each other, indicating a common ancestor. The results revealed that C. dentatus discriminated from the species with n = 10 chromosomes, C. boissieri and C. glaucus assigned in to separate subsection. In summary, this study has shown that domesticated and wild genotypes were clustered into two major groups indicating these markers as appropriate tools to amassment genetic diversity and genome mapping.