The purpose of this study was to investigate the utility of the different types of molecular markers in assessing the genetic diversity of seven Serbian broomrape (Orobanche cumana Wallr.) populations, such as ISSR and SSR. The discriminatory potential of SSR markers was on average lower (Rp = 2.46) than for ISSR (Rp = 13.85), indicating a large interpopulation genetic variability. According to the indices of genetic diversity, a higher intrapopulation molecular variability in the case of SSR markers (PIC = 0.58; H = 0.63) compared to ISSR (PIC = 0.33) was revealed. AMOVA analysis also showed a high genetic diversity within populations with SSR markers (within pops 53% and among pops 47%) and the most genetic diversity among populations with ISSR (34% and 66%, respectively). These results showed a high degree of genetic variations among and within broomrape genotypes from Serbia which favor the evolution of more virulent physiological races of O. cumana. This study can serve as scientific support for future researches in monitoring and developing strategies to improve sunflower crops resistant to this pathogen.