Cistus heterophyllus subsp. carthaginensis is a critically endangered plant taxon from E Spain. Only two populations, one in La Pobla de Vallbona, Valencian Community (consisting of only one plant) and the other in Llano de Beal, Cartagena municipality, Autonomous Community of the Region of Murcia, are presently known. The low number of individuals and introgression by the closely related C. albidus are the major threats to the conservation of Cistus heterophyllus subsp. carthaginensis. In the years 2007, 2008, 2011, and 2013 seeds were collected from the Murcia population within the efforts to protect this taxon. In this work, we compare the natural population of Murcia with the ex situ plants originated from the recollected seeds, the only known sample from Valencia, and a specimen of North Africa of C. heterophyllus using genotyping by sequencing. The natural population of Murcia shows clear signals of introgression by C. albidus while the ex situ plants are much less affected, suggesting that the major part of the introgression of the natural populations took place after the specimens for the ex situ collection were taken. The Murcian samples seem not to be very close to the Valencian plant, but the systematic relationships among the studied populations remain unclear. The ex situ conservation efforts are a key to the conservation of this taxon.