This study aimed to assess the genetic diversity and population structure of 18 sugar beet (Beta vulgaris L.) genotypes using ISSR molecular markers. Initially, 18 ISSR primers were screened, of which six were selected based on their polymorphism and amplification efficiency. A total of 41 bands were scored, 29 of which were polymorphic, resulting in a polymorphism rate of 75.83%. Genetic similarity coefficients calculated using Jaccard’s index were used to generate a UPGMA dendrogram, revealing both closely related and highly divergent genotypes. Principal Component Analysis (PCA) confirmed these results, indicating a complex genetic structure with potential heterotic pools. genotypes SB-7 and SB-17, which showed the highest similarity (0.806) in the UPGMA dendrogram, were also positioned in close proximity on the PCA plot. In contrast, genotypes SB-2 and SB-15, which demonstrated the lowest genetic similarity (0.461), occupied distant locations along the principal axes, reinforcing their genetic divergence. STRUCTURE software was employed to analyze population structure, showing that some genotypes exhibited high genetic purity while others displayed admixture. The findings underscore the effectiveness of ISSR markers in evaluating genetic variation and provide valuable insights for future sugar beet breeding programs aimed at improving adaptability, productivity, and genetic resource conservation.