In this study, a total of 19 retrotransposon-based iPBS markers were used to determine the genetic relationship among 11 chestnut cultivars (Castanea sativa Mill.) that are important in Turkey. In the study, chestnut cultivars named Hacıömer, Osmanoğlu, Sarıaşlama, Erfelek, Kemer, Işıklar, Şekerci, Siyah Bursa, Tülü, Bouche De Betizac and Marigoule were preferred. The allele profiles obtained from PCR reactions and capillary electrophoresis system were evaluated. Size ranges of alleles, number of average allele, number of total allele, number of polymorphic alleles, polymorphism rate, mean heterozygosity, polymorphism information content, marker index and discriminating power were determined at a successful level. In addition, UPGMA dendograms were created and principle coordinate analyzes were performed. According to UPGMA dendogram, chestnut cultivars of Hacıömer and Şekerci were determined as the most similar cultivars with a similarity coefficient value of 0.924 and they formed a subgroup together with the chestnut cultivars of Osmanoğlu and Erfelek which showed close similarity with these two cultivars. On the other hand, it was concluded that two chestnut cultivars, Işıklar and Şekerci, which are highly similar to each other by some sources, are very different from each other. Besides, the use of retrotransposon based iPBS markers in chestnuts in Turkey was made for the first time in this study, and it has been proven that the genetic relationship among chestnut cultivars can be determined with high accuracy. It has been determined that these markers will be useful for molecular characterization studies in plant breeding programs.