Crocus sativus L., saffron Crocus, is a sterile triploid (2n=3x=24) species of unknown relationship to other diploid and polyploid species in the genus Crocus (Iridaceae). The species have large genomes (typically 3000Mbp 1C), much repetitive DNA, and show high morphological diversity within and between species, with no clear phylogenetic patterns below the level of section Crocus, series Crocus. I aimed to examine molecular diversity in C. sativus and related species by analysis of sequences and IRAPs (Inter Retroelement Amplified Polymorphisms). Repetitive DNA sequences and genomic DNA from various species were used for in situ hybridization, with chromosome morphology, to infer relationships and ancestry of saffron. The IRAP analysis, involving 63 primer combinations and 4745 polymorphic bands, revealed no polymorphism within 17 saffron accessions obtained from across the world from Kashmir through Iran to Spain. In contrast, high levels of polymorphism were identified between accessions of six wild Crocus series Crocus species, with further variation between the species. Analysis of 123 sequences of the ATP-synthase gene and 107 TC25 gene-SSR sequences from seven saffron accessions and eight wild species showed that the saffron accessions often carried three alleles, a result also found with clustering of published EST sequences. The analysis showed many alleles were shared by Crocus species and did enable a well-resolved phylogeny. Chromosome analysis grouped saffron chromosomes into 8 groups of 3, but one chromosome differed from the other two. It was concluded 1) Saffron crocus has minimal genotypic variation and the triploid hybrid species is most likely to have arisen only once; 2) Saffron is a allotriploid species, with the most likely ancestors being C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics and show the need for conservation and collection of wild Crocus.