In the current study, a total of 235 genotypes of Brachypodium distachyon (BD) were studied and targeted to evaluate the genetic diversity, population structure and relative kinship of these genotypes using inter-primer binding site (iPBS) markers. Twenty-eight iPBS markers were used, and 184 clear and sharp polymorphic bands were produced. The polymorphism information content and diversity parameters indicated the existence of an appropriate amount of genetic diversity in the analysed genotypes. The results of the dendrogram with heat map and principal component analysis (PCoA) revealed that 235 BD genotypes were grouped into two separate clusters. The population structure was calculated using the STRUCTURE software, and two major sub-groups (K = 2) were established. A total of 95.1% of the relative kinship estimates being less than 0.4 for all genotypes. The findings of this study concluded that iPBS markers are highly polymorphic and are very efficient in the evaluation of the genetic diversity of B. distachyon.