This report describes a new, sensitive and specific protocol for rapid detection and quantification of Yersinia enterocolitica in artificially contaminated raw milk samples. The new method is based on an optimized real-time PCR protocol with a TaqMan probe. The primers and probe are based on the chromosomal ail gene. This method was successful for both intended uses: (1) direct detection and quantification of Y. enterocolitica in artificially and naturally contaminated raw milk samples and (2) characterization of growth potential of different serotypes of Y. enterocolitica in raw milk at the most commonly used storage temperatures. The recent method eliminates the pre-PCR enrichment step, which makes it possible to quickly assess milk-related consumer exposure to this pathogen.