Wild sunflower species are adapted to diverse habitats and possess considerable variability for biotic and abiotic traits, therefore wild germplasm can be a real source for improving the narrow genetic base of the cultivated sunflower crop by introgression of new genes which were previously identified in wild genotypes. In order to effectively exploit these genes in cultivated genotypes, researchers need more information regarding the quantity and distribution of the genetic variability available within the wild sunflower species, which can offer a multitudine of valuable traits for traditional or molecular breeding. This study was conducted to reveal the genetic diversity of wild and cultivated sunflower genotypes using ISSR markers. From a total of 19 ISSR markers, which were used to evaluate the genetic diversity, eight markers (UBC808, UBC823, UBC834, UBC836, UBC840, UBC845, UBC853, 17899A) clearly differentiated the wild sunflower genotypes from the wild sunflower and/or the cultivated sunflower. The primers amplified a total of 120 alleles ranging from 10 to 22 alleles per marker. A wide range of fragment length was detected among the accessions, from 140 to over 1500 bp. The neighbor-joining dendrogram, based on Rogers genetic distance, of the genotypes studied consisted of two main clusters of different sizes: five entries were grouped into cluster A and the remaining six entries were grouped into cluster B. It is interesting that genotypes of Helianthus anuus L., Helianthus maximiliani and Helianthus argophyllus were clustered together. Another aspect observed, refers to interspecific variability for Helianthus debilis genotypes. Regarding the PCA, the first two principal axes accounted for 26.8% (CP1) and 16.1% (CP2) of the total variation, respectively, together explaining 42.9% of the total variability. In the future, this molecular genetic information can be combined with morphological and biochemical data to improve the sunflower breeding program.