Wild species of safflower, Carthamus oxyacanthus Bieb., is highly crossable with cultivated species, C. tinctorius L. and could be directly exploited in broadening safflower gene pool and improving the crop for biotic and abiotic stress environments. In this study, genetic diversity among accessions of C. oxyacanthus and their relationships with cultivated safflower were evaluated using agro-morphological traits and polymorphic inter-simple sequence repeats (ISSR) markers. Significant variation was observed among accessions particularly for seeds per capitulum, seed yield per plant, harvest index and capitula per plant. Cluster analysis based on agro-morphological traits classified the wild accessions in two groups according to their geographical regions, and separated them from the cultivated genotypes. ISSR marker also revealed a high genetic variation among the accessions, and cluster analysis based on this marker divided genotypes into four groups, with cultivated ones in a separate clade. Genetic variation observed among the wild safflower germplasm at the DNA level was higher than the agro-morphological traits, indicating that ISSR is an effective marker system for detecting diversity among safflower genotypes and their genetic relationships. Accessions of C. oxyacanthus with high genetic relationship to cultivated species could be used for interspecific hybridization in breeding programs of safflower.