The stone crayfish is a native European freshwater species with significant population declines caused by anthropogenic pressure onto its habitats, climate change and spreading of non-indigenous invasive crayfish and their pathogens (e.g., Aphanomyces astaci). We present the first record of an Austropotamobius torrentium population in a small stream from the southern lowlands of Romania, in an area without limestone or karst, known as a typical stone crayfish habitat in this country. No additional populations were found in the neighbouring streams. The mtDNA analysis based on sequencing a short fragment of the cytochrome c oxidase subunit I (COI) gene revealed that specimens from this newly discovered population belongs to the most widespread phylogroup of A. torrentium (CSE) and share the same haplotype as the specimens found in Croatia. Unfortunately, this crayfish population has disappeared most likely because of the crayfish plague since pathogen A. astaci was isolated from moribund crayfish. Our study highlights the vulnerability of isolated relic populations in the face of outside stressors.