Saffron crocus (Crocus sativus) is the source of the most expensive spice of the world, produced from manually harvested stigmas, thus serving as a cash crop for rural communities. However, despite its economic importance, its genome and chromosomes are poorly studied. C. sativus is a sterile triploid species harboring eight chromosome triplets, and propagated only as a clonal lineage by corms. Saffron's evolutionary origin, parental species and allo- or autotriploidy has been a matter of discussion for almost a century. We performed a survey sequencing of the saffron genome and selected cytogenetic landmark sequences consisting of major tandem repeats, which we used as probes in comparative multicolor fluorescent in situ hybridization (FISH). We tagged 92 chromosomal positions and resolved the chromosomal composition of saffron triplets. By comparative FISH of six Crocus species from 11 accessions, we demonstrate that C. sativus is an autotriploid hybrid derived from heterogeneous Crocus cartwrightianus cytotypes. The FISH reference karyotype of saffron is crucial for integrating genome sequencing data with chromosomes and for investigating the relationship among Crocus species. We provide an evolutionary model of the saffron emergence; the knowledge of the parental origin offers a route towards the resynthesis of C. sativus from C. cartwrightianus to broaden saffron's gene pool.