The multipronged nuclear DNA assay by random amplified polymorphic DNA (RAPD) fingerprinting, ribosomal DNA repeat unit length polymorphism, internal transcribed sequence (ITS) RFLP, and comparative sequence analysis of ITS and external transcribed sequence (ETS) regions of the 29 accessions belonging to 18 Carthamus taxa including five unverified species was undertaken to obtain new information on (1) interrelationships among botanical varieties of cultivated safflower, C. tinctorius, and phylogenetic relationships (2) among the safflower and its close relatives and (3) that of Carthamus species and subspecies. The root tip cells of the 12 accessions contained 24 chromosomes followed by 64, 44 and 20 chromosomes in 9, 6 and 2 accessions, respectively. Barring C. lanatus, the accessions within each taxon had the same zygotic number. The present results strongly support the view that the wild C. palaestinus (2n = 24) and the cultivated C. tinctorius (2n = 24) are closely related. With few exceptions, all DNA based dendrograms support three lineages within the genus. One lineage is constituted by C. arborescens (2n = 24) alone. The present data indicates that because of unique composition of its nuclear genome vis-à-vis other Carthamus taxa, C. arborescens should be placed in a separate subgenus. The two remaining lineages, constituted by the taxa with 2n = 24, and the taxa with 2n = 20, 2n = 44 and 2n = 64, respectively should be given the rank of two taxonomic sections in the other subgenus. The present study also demonstrates that none of the present taxa with 2n = 24 have contributed to the origin of polyploid taxa. Carthamus boisserii (2n = 20) and C. glaucus ssp. anatolicus (2n = 20) are more likely to be one of the diploid progenitor of C. lanatus ssp. creticus (2n = 64), C. lanatus (2n = 44), C. lanatus ssp. lanatus (2n = 44) and C. lanatus ssp. montanus (2n = 44), and C. lanatus ssp. turkestanicus (2n = 64), respectively. Within Lanatus species complex, constituted by C. lanatus, C. lanatus ssp. lanatus, C. lanatus ssp. montanus, C. lanatus ssp. turkestanicus and C. lanatus ssp. creticus, high proportion of autapomorphic characters and low number of synapomorphies in the ITS and ETS sequences suggest a relatively recent diversification of the taxa within the species complex. Carthamus lanatus ssp. creticus (2n = 64) and C. lanatus ssp. turkestanicus (2n = 64) within the complex deserve species rank. This analysis provided evolutionary relatedness of the five unverified taxa with the known Carthamus taxa.