Saffron as the most expensive spice in the world is obtained from dried stigmas of Crocus sativus L. flowers and used mainly as food coloring and flavoring in food industry and also the use of its effective components in medicine. In this study genetic diversity among 28 saffron genotypes collected from different regions of Iran was evaluated using sequence related amplified polymorphism (SRAP) marker. Nineteen SRAP primer combinations amplified a total of 147 polymorphic fragments with an average of 7.74 fragments per each primer combination and average of polymorphic information content (PIC) of 0.15. Cluster analysis using NJ (Neighbor-Joining) method was divided the genotypes into four groups that most of them clustered in a major group. Principal component analysis (PCA) showed that cluster analysis is more appropriate for revealing genetic relationship of saffron genotypes. The close relationships among saffron genotypes revealed in this study can be due to vegetative propagation, human selection of superior genotypes and existence of narrow genetic base of saffron. The results confirmed that the SRAP markers don’t have ability for discrimination of genetic diversity among saffron genotypes. In order to design specific primers for amplification of saffron DNA, we used RAPD markers. Twenty five random primers were tested, among them 6 primers produced bands with different size and appropriate amplification. Specific bands were cloned for sequencing and designed specific primers. After optimization of PCR conditions for designed specific primers, they were tested with mixed of saffron, safflower and corn DNAs as misused saffron. The results of these experiments confirmed the specific amplification of saffron. Specific primer was also designed and tested for safflower using ITS sequences and the results revealed specific amplification of safflower under mixed saffron and safflower. Since the results of PCR is expressed as qualitative (presence or absence bands), for quantification of saffron in misused samples, we used Real-Time PCR reaction using designed saffron specific primers based on DNA fragments of RAPD marker and using sybergreen kit. Because of unknown reign of fragment amplified with RAPD primers and high sensitive Real-Time PCR reaction to noecific amplification in low values and also low noecific amplification in sybergreen method, the results in Real-Time PCR reaction were not reliable. There for, designing of specific primers from coding region of saffron genome for specific amplification in Real Time PCR and quantification of saffron in misused samples were recommended.