This study aimed to evaluate the genetic diversity of soybean genotypes using ISSR molecular markers within the scope of soybean breeding studies conducted at the Faculty of Agricultural Sciences and Technology, Sivas University of Science and Technology. A total of 18 soybean (Glycine max (L.) Merr.) genotypes, including 13 soybean lines obtained through hybridizations performed in accordance with breeding objectives and 5 commercial cultivars, were analyzed. Using seven ISSR primers, a total of 78 DNA bands were obtained, of which 32 were identified as polymorphic, corresponding to a polymorphism rate of 41.02%. These findings revealed moderate genetic variation among genotypes and confirmed the applicability of ISSR markers for the analysis of soybean genetic resources. According to the UPGMA clustering analysis, the genotypes were divided into two main groups; genotypes S-6 and S-7 exhibited the highest genetic similarity (98.4%), whereas genotypes S-3 and S-13 showed the lowest similarity (79.3%). Principal Component Analysis (PCA) supported the genetic structure obtained from UPGMA and enabled the multidimensional visualization of relationships among the genotypes. The distribution of commercial cultivars in different clusters indicated that these cultivars possess distinct genetic backgrounds. The results demonstrate that ISSR markers are effective, reproducible, and cost-efficient molecular tools for distinguishing genetic similarities and differences. This study provides important scientific data for parent selection in soybean breeding programs, identification of heterotic combinations, and sustainable management of genetic resources.