Rhinolophus mehelyi (Mehely's horseshoe bat) is a vulnerable species with an increasingly fragmented distribution. In Romania, populations of R. mehelyi have experienced a dramatic decline over the past 50 years, and the current population size is estimated at only 100 adult individuals inhabiting almost exclusively the Limanu cave. In the present study, we investigated the genetic consequences of population decline for the viability of the remaining population of R. mehelyi in Romania. We sequenced and analyzed a 359-bp fragment of the mitochondrial control region from the only known Romanian population and compared it with two geographically close colonies from Bulgaria. A single haplotype was found in the Romanian population compared to 10 in the Bulgarian population, suggesting genetic isolation.