Knowledge of genetic diversity is one of the most essential tasks for sustainable management of medicinal plants. In the present study, the molecular variability and population structure in a set of Ferula assafoetida accessions collected from diferent geographical regions of Iran were evaluated using start codon targeted polymorphism (SCoT) and universal rice primer (URP) markers. Based on results, SCoT (12 primers) and URP (12 primers) markers amplifed 192 and 149 polymorphic fragments, respectively. The polymorphism information content (PIC) estimated by URP and SCoT ranged from 0.31 to 0.43, and 0.34 to 0.44, respectively. The analysis of molecular variance revealed the greater genetic variation within population compared to among them. The Kerman’s population showed the highest values for the heterozygosity (He), percentage of polymorphic bands (PPB), Shannon’s information index (I), and efective number of alleles (Ne). The neighbor joining (NJ) clustering algorithm grouped all investigated accessions into six and fve main groups based on URP and SCoT data, respectively. The results of structure analysis based on both marker systems showed that all accessions were divided into fve subpopulations. Furthermore, principal coordinate analysis (PCoA) supported these fndings. In conclusion, our results revealed a high level of genetic diversity and gene fow between F. assafoetida populations. Moreover, the SCoT markers were more efective than URP in detecting the genetic diversity in F. assafoetida populations. Hence, the use of SCoT markers in further genetic research such as genetic fngerprinting and association analysis would be recommended
