Therefore, the aim of our study was to develop a multiplex PCR assay for efficient and quick detection of L. delbrueckii subsp. bulgaricus. Our methodology was based on the tuf gene sequence polymorphism. Polymorphic sites within the tuf gene have been used successfully as a molecular marker ( Sheu et al., 2009). It is worth to mention that this gene displays less sequence homology in comparison to other ubiquitous genes used in the past, thus it has higher discrimination potential