The Blasius's horseshoe bat (Rhinolophus blasii) shows a fragmented distribution in the European part of its range, and its populations are threatened by many human-induced factors. The status and distribution of the species in Romania are largely unknown. In recent years, regular bat surveys and monitoring have confirmed the presence of the species in north-western Romania, in Gălăşeni Cave, Pădurea Craiului Mountains, representing the northernmost distribution point of R. blasii in Europe. In our study we investigated whether this northernmost colony is genetically isolated from, or related to, larger populations in south-western Romania. A combined analysis of two mitochondrial (12S, HVII) and two nuclear (RAG2, DBY7) genetic markers was used in order to reveal possible relationships between these populations. The small number of haplotypes, the presence of shared haplotypes and overall low population structure suggest that the south-western and north-western Romanian populations are related through yet unidentified colonies of R. blasii, with continuous migration between them. Our results also indicate that the R. blasii colony from Gălăşeni Cave was established by female individuals migrating not far from their birthplace. To preserve this marginal population in a long term, conservation measures need to be strengthened and properly enforced, in parallel with the search for new R. blasii colonies in the region. To our knowledge, the present work represents the only molecular study of R. blasii in Europe.