In conclusion, in this study we presented a new multiplex PCR-based assay for efficient identification of L. delbrueckii subsp. bulgaricus in a single reaction. Our method achieved selective differentiation of L. delbrueckii subsp. bulgaricus even among very closely-related subspecies, including L. delbrueckii subsp. delbrueckii and L. delbrueckii subsp. lactis and proved to be an efficient tool for rapid and reliable verification of the presence of L. delbrueckii subsp. bulgaricus in yoghurt that could be valuable in the food industry.