Orobanche cumana, commonly known as sunflower broomrape, poses a significant risk to sunflower cultivation in areas located along the Black Sea and across Europe. A study was conducted to analyze the genetic diversity and differentiation among populations of this parasitic plant originating from Bulgaria, Turkey, Republic of Moldova, and Romania. A total of 269 individuals from 23 populations were genotyped using 13 ISSR markers. The frequency distribution of alleles in the broomrape populations from Romania, Turkey and Bulgaria is more homogeneous than that from Moldavian populations. However, according to genetic diversity parameters O. cumana populations from Moldova and Turkey were more variable (total gene diversity Ht=0.188; 0.214), than those from Bulgaria and Romania (Ht=0.112; 0.067). The highest values of gene diversity within populations (Hs = 0.138) were found in Turkish broomrape. The value of Gst ranged between 0.359-0.516, indicating a very high level of genetic differentiation among populations. These results are consistent with low Nm values (0.468-0.895). Pairwise differentiation index (PhiPT) and Nei's unbiased measures of genetic distance (GD) showed similar patterns, indicating a maximum (PhiPT=0.500; GD=0.261) and, respectively, minimum (PhiPT=0.238; GD=0.079) values between broomrape from Turkey and Romania, respectively, Romania and Moldova. Dendrogram constructed using the UPMGA method based on Nei's genetic distances and Pearson's dissimilarity clustered together Moldavian and Romanian broomrape and grouped in two separate clusters populations belonging to Bulgaria and Turkey. The PCA analysis confirmed the results of UPGMA clusterization. Overall, both methods suggest that the groupings of broomrape are partly determined by its geographical origin, as well as by the genetic differences and similarities accumulated over time, and are not related to virulence.