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As tomatoes are one of the most important vegetable crops, the breeding of new cultivars is of major importance in order to respond to the rising demands of consumers and producers. The aim of the study was to test if the ISSR marker system is a viable tool for characterizing the genetic variability in genotypes, used in a breeding program aimed at improving the fruit antioxidant contents. Eight genotypes with varying levels of antioxidant content in the fruit were examined. The results we obtained from our analysis show that the ISSR marker system could be successfully applied for the needs of breeding programs, seed production and genotyping of existing and newly created tomato cultivars.