Saffron (Crocus sativus L.) is one of the most valuable medicinal and spice herbs in the world. In spite of the ancient cultivation history in Iran, there are limited breeding studies on the plant due to its vegetative reproduction. In order to evaluation genetic diversity of Iranian saffron germplasm, sixty-five different saffron accessions from the main cultivation areas in Khorasan including Torbat heidarieh, Gonabad, Mahvelat, Ghaenat and Ferdows were collected and were studied by molecular markers. The used RAPD and ISSR primers produced 43 and 122 polymorphic markers loci, respectively, and totally 165 markers with average of 7.5 markers by each primer, totally. Diversity index ranged from 0.36 to 0.7 with average of 0.23. Also, marker index with the average of 0.16 varied in the range of 0.2 to 0.7. The accessions from Ghaenat and Mahvelat had the maximum (83.03%) and the minimum (52.73%) polymorphism, respectively. Grouping the studied saffron accessions using cluster analysis displayed four distinct groups which had little correspondence to their collection areas, while clustering for the main cultivation areas had relatively good correspondence to their geographical distances. So, it is expected to have nearly approaching improvements of qualitative and quantitative yields via the selection of superior clones of saffron. Key words: Saffron, Molecular variation, Germplasm, RAPD, ISSR, Khorasan region, clustering