Saffron (Crocus L.) is a member of Crocoideae, the biggest of four subfamilies in the Iridaceae family. It has 2n = 3x = 24 chromosomes and is triploid; thus, it is sterile. In previous research, different molecular DNA markers were used but molecular characterization and genetic diversity of this complex genus have not yet been clarified. Therefore, current study aimed to determine the molecular characterization of saffron and its close relative species using inter-primer binding site (iPBS)-retrotransposon markers. Eighty-three iPBS-retrotransposon primers were used in 28 C. sativus genotypes and 17 close relative species of saffron to identify their genetic diversity. Sixteen polymorphic iPBS-retrotransposon primers generated a total of 401 polymorphic scorable bands. The mean PIC value, Nei's genetic diversity and Shannon's information index (I) were calculated as 0.85, 0.16 and 0.29, respectively. The results of the Unweighted Pair Group Method with Arithmetic mean UPGMA dendrogram and Principal Coordinates Analysis PCoA analysis indicated a spatial representation of the relative genetic distances among 28 saffron samples and the 17 close relative species were categorized under two distinct groups. Saffron genotypes showed very limited genetic variation and according to the iPBS-retrotransposon data, its close relatives were C. cartwrightianus and C. pallasii subsp. pallasii.