Technical Abstract: It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, because those diseases can be extremely similar in the early stages of their pathogenicity. In this study, we report the development and validation of a real-time RT-PCR assay using a Taqman labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5’ UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as 2 turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Validation of the assay was completed with 1,329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds, were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and tested them by the real-time RT-PCR assay to monitor viral load during the course of infection. We found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 28 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83% respectively indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other upper-respiratory disease agents of global importance. The test was extremely sensitive and specific, and can be used to quantify viral RNA in clinical samples.