Orobanche cumana is found in the Iberian Peninsula as an allochthonous species parasitizing exclusively sunflower in contrast to the closely related species Orobanche cernua which is an autochthonous species that only parasitizes wild Asteraceae hosts. Ten O. cumana populations were collected in the two traditional areas of sunflower broomrape occurrence the Guadalquivir Valley Southern Spain (six populations) and Cuenca province Central Spain (four populations). Twelve O. cernua populations were collected on wild hosts across its natural distribution area in Southeastern Spain. Genetic relationships within and between both sets of populations were studied using a set of 50 robust and co-dominant SSR markers from O. cumana . The results supported the taxonomic separation of the two species and the existence of two distant genetic groups for O. cumana one in Guadalquivir Valley and another one in Cuenca province. The inter- and intra-population variability was extremely low for O. cumana whereas the overall genetic diversity was much higher for O. cernua . The genetic structure of O. cumana populations probably reflects a founder effect with the two genetically distant groups deriving from separate introduction events. The high degree of genetic differentiation observed in O. cernua is mainly explained on the basis of restricted gene flow due to ecological barriers together with the occurrence of a predominantly self-pollinating mating system. Complementary diversity studies on both species in its current distribution area are required for understanding global genetic variability and evolutionary characteristics of the parasitism.