The genetic diversity of 4 broomrape populations of Debovo, Selanovtsi, Radnevo and Rosenova from Bulgaria was evaluated using inter simple sequence repeat (ISSR) markers. Analysis of genetic structure of populations revealed the average observed number of alleles (Na=1.4581) and effective number of alleles (Ne=1.2179). The average calculated value of Nei’s gene diversity (H=0.1368), Shannon’s Information index (I=0.2132), number of polymorphic loci (NPL=62.75) and percentage of polymorphic loci (PPL=45.81) indicated a medium level of genetic variation exists among the broomrape accessions in the studying populations. The highest level of diversity was found in population from Debovo, followed by Radnevo, Rosenova and Selanovtsi. Based on clustering method, a Ward’s tree was constructed and grouped the entire genotypes into 3 major clusters. PCA analysis revealed that first principal component extracted maximum variation (F1=27.27%) than second principal component (F2=17.39%). Both of the methods (AHC and PCA) distributed the 48 broomrape accessions in three groups and presented similar grouping of the genotypes. AMOVA test demonstrated the higher genetic diferences among populations (53%) and lower level (47%) within the populations. The total genetic diversity (Ht=0.2700) and genetic diversity within populations (Hs=0.1525) were no significantly that reflect low population heterozygosity. It was revealed a great genetic differentiation (Gst=0.4354) with intermediate gene flow (Nm=0.6485) among the broomrape populations. All results showed that there is medium level of diversity within populations and large differentiation among populations that suggest the important role of genetic drift in genetic structure differentiation among broomrape populations. Genetic relatedness and population structure are essential condition for wider natural distribution, stronger environmental adaptability, survivability and evolutionary potential of a species in changing environmental conditions [1]. Evaluation of genetic diversity of the parasitic species O. cumana is important tool in germplasm characterization and this information may be highly relevant to contribute for the development of sustainable control strategies of pathogen and breeding programs of sunflower resistance to broomrape. This investigation aimed to detect and characterize the genetic variability and similarity in O. cumana populations collected from naturally Orobanche-infested fields in different regions of Bulgaria using ISSR molecular markers.