Durum wheat is important cereal that is widely cultivated all over the world. In this study, genetic diversity in a mini core collection of the 43 durum wheat genotypes (including 25 breeding lines and 18 landraces) was investigated using fifteen inter-simple sequence repeat markers (ISSR). Based on the results, high levels of polymorphism (98.70%) were observed, which indicated that ISSR was efficient technique to detect the genetic variation of this collection. Analysis of molecular variance (AMOVA) revealed high genetic variability (90%) within breeding lines and landrace genotypes. Comparing the genetic diversity of breeding lines and landraces, based on genetic variation parameters, revealed that the effective number of alleles (Ne), Nei's gene diversity (H) and Shannon's information index (I) in the landrace genotypes is higher than breeding lines. Cluster analysis classified all individuals in five groups. Also, inter-population differentiation (Gst) estimated on the basis of these primers represented that, 93% of the total genetic variability refer to variation within two sets of genotypes. Overall, our results revealed a high level of genetic variation among a mini core collection of durum wheat, especially Iranian landraces, which can be of interest for conservation and future breeding programs.