In this report, we describe the development and evaluation of a 5' nuclease PCR assay for the detection of pathogenic Yersinia enterocolitica (YE). The assay targets the chromosomally encoded invasion gene ail. Three different primer/probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. The TM1 set displayed the highest specificity, accurately detecting each of the 26 YE strains and none of the 21 non-enterocolitica strains. This set was sensitive to approximately 0.5 pg of purified Y. enterocolitica DNA. The TM2 set was the most sensitive, allowing detection in the range of 0.25 pg of purified DNA. However, it was not specific and failed to recognize 10 of the YE strains used in this study. Sensitivities comparable to TM1 were achieved with the TM3 set; cross-reaction with non-enterocolitica strains was not observed. However, this set failed to positively identify all of the YE strains tested.