Verbascum (Mullein) flowers are highly valued as natural remedy for various respiratory diseases. Verbascum anisophyllum Murb. is a Balkan endemic, protected by law and included in the Bulgarian Red Data Book as "Critically Endangered". Thus, a strict conservation policy and a reliable evaluation of its genetic resources are required, considering its narrow distribution range and the increasing risk from destruction of its habitats. Here, we used Inter-simple sequence repeat (ISSR) markers to characterize the genetic diversity and to assess the genetic differentiation between the existing populations of VerbascUM anisophyllum in Bulgaria. The level of genetic diversity found herein clearly indicates a long-term potential for adaptability of this endangered plant. Our findings provide important knowledge of population genetic structure of this species, thus representing a strategy for its efficient conservation and utilization.