Since floras of oceanic islands are distinct from those of the closest continents, islands can serve as useful systems for understanding the evolution of host specificity during speciation of parasitic plants. Orobanche boninsimae (Orobanchaceae) is a holoparasitic plant species endemic to the isolated Bonin (Ogasawara) Islands; previous observations suggested several possible host species for O. boninsimae. In this study we sought to provide more definitive identification of O. boninsimae host plants through DNA analyses of host plant roots. We investigated nine populations of O. boninsimae from Chichijima and Hahajima Islands and determined their hosts by DNA barcoding of the chloroplast trnH-psbA region. We determined that three endemic woody species, Ochrosia nakaiana (Apocynaceae), Melicope grisea (Rutaceae), and Melicope nishimurae (Rutaceae), and one exotic tree, Bischofia javanica (Phyllanthaceae), were host species of O. boninsimae. These three families are newly recorded as hosts for the genus Orobanche, suggesting that O. boninsimae might have developed these unique host specificities in the islands. In addition, we established that O. boninsimae parasitizes completely different species between the islands, even though all available host species of O. boninsimae execpt for M. nishimurae were commonly distributed throughout these islands. This result suggests that intraspecific differentiation for host preference and possibly cryptic speciation has occurred between isolated islands.