A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Yersinia enterocolitica. The assay used specifically designed primers to target within the phoP gene and correctly identified all 37 strains of Y. enterocolitica and 50 non-Y. enterocolitica strains. The probability of detection was 100%, when the DNA of extracted from 10(1) CFU Y. enterocolitica was used as template in LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in Luria-Bertani broth, followed by extraction and purification of DNA. In this way, 102 various food samples were investigated for Y. enterocolitica including 79 minced pork samples and 23 powdered milk samples. The accuracy of LAMP was shown to be 100% when compared to the standard method, ISO 10273. This combination of sample enrichment, and LAMP assay can detect 2.2 CFU per 100 g food samples. The overall analysis time for the LAMP assay was approximately 24 h. This is in contrast to 5 days of analysis time required for the traditional culture method. Consequently, the LAMP described here, has the potential to become a standardized method for the rapid detection of Y. enterocolitica in diagnostic laboratories once further validated by inter-laboratory studies.