Broomrapes (Orobanche L.) are holoparasitic plants, parasitizing roots of a wide range of host plants. In this study, genetic polymorphism among 44 Orobanche aegyptiaca Pers. individuals collected from different regions of northwest Iran was investigated using inter-simple sequence repeat (ISSR) markers. Two hundred-sixty one discernible bands were amplified using 20 ISSR primers which 245 (94%) was polymorphic, indicating considerable genetic variation among the examined individuals. The number of polymorphic bands per primer ranged from 4 to 17, averaging 12.25. UPGMA clustering using Jaccard's similarity coefficients revealed six main groups. Genetic similarity coefficients varied from 0.71 (between individuals 23 and 27) to 0.34 (between 13 and 30). A model-based Bayesian approach subdivided 38 out of 44 broomrape genotypes into 2 genetic groups and the remaining ones were categorized as mixed genotypes based on Q values. According to an analysis of molecular variance, 99% of the total variation was partitioned within genetic groups. The results demonstrated the potential usefulness of ISSR markers for determination of genetic variation in O. aegyptiaca. Understanding the variability in broomrape is important when attempting to develop resistant host crops against this parasite.