Marker-assisted breeding serves as a potent tool for screening target germplasm, assessing genetic diversity, and determining breeding potential of a crop. Therefore, inter primer binding site (iPBS)-retrotransposons marker system was employed to evaluate a collection of 33 Brassica genotypes, including 10 Brassica juncea, 5 B. oleracea, 7 Sinapis alba, 5 B. nigra, and 6 B. rapa, were utilized to evaluate their genetic diversity and variations 10 polymorphic primers that generated a total of 144 bands. Various diversity indices were calculated in the studied germplasm, including polymorphism information content (0.13–0.30), effective number of alleles (1.217–1.689), Shannon’s information index (0.244–0.531), and gene diversity (0.148–0.370). These indices collectively affirmed substantial genetic variations within the germplasm. Molecular variance analysis revealed that the majority (62%) of genetic variations were present within populations. The Brassica accessions were categorized into three populations utilizing a model-based structure algorithm. Evaluation of diversity indices based on the structure indicated that populations III and II exhibited higher diversity. Principal coordinate analysis and neighbor-joining analysis further corroborated the three distinct populations, confirming the reliability of the STRUCTURE analysis. Notably, the genetic distance assessment identified BN1 and BN3 from B. nigra species and the genotypes BO1 and BO3 from B. oleracea as genetically diverse mustard accessions. The extensive genetic diversity observed within the Brassica germplasm underscores its significance as a valuable genetic resource for comprehensive Brassica breeding programs. Moreover, these accessions hold promise as suitable candidates for heterosis breeding initiatives aimed at improving mustard production.