Ramonda serbica Panč. (Gesneriaceae) is an endangered endemic species of the Balkan Peninsula. It has been the subject of several studies in the past, but has not yet been investigated in terms of its genetic variation. Here, an Inter Simple Sequence Repeat (ISSR) analysis was performed to determine the genetic diversity of five populations sampled from the geographically eastern fringe of its range in northwestern Bulgaria. We found low levels of genetic diversity and significant genetic differentiation among the investigated populations, typical of a leading edge scenario. From a conservation point of view, the low genetic diversity, together with the presence of only few extant localities, underline the need for urgent in situ and ex situ conservation actions to ensure the long-term presence of R. serbica in Bulgaria.