AimsThe aim of the study was to develop a multiplex real-time PCR method to identify Campylobacter jejuni containing mutations commonly associated with macrolide resistance. Methods and ResultsA multiplex fluorescence real-time PCR assay was developed based on TaqMan minor groove binder (MGB) probes. The VS1-MGB probe was designed based on the VS1 gene and was used to identify Camp.jejuni. The 23S rDNA-MGB probe was designed to distinguish macrolide resistance mutations in 23S rDNA, while 57D-MGB and 74D-MGB were designed to detect resistance mutations in ribosomal protein L4. The specificity and accuracy of our method were identical to the conventional biochemical tests, mapA PCR, minimum inhibitory concentration (MIC) determination and DNA sequencing. The linear detection limit of the method was 003ng genomic DNA and three colony formation unit (CFU) per reaction. In 6 of 18 cases, the nature of Erythromycin resistance could be correctly determined from natural isolates; absence of the tested mutations was demonstrated in the remaining four resistant isolates. ConclusionsA multiplex TaqMan MGB real-time PCR assay with high specificity and accuracy was developed tosimultaneously identify Camp.jejuni and detect the gene mutations associated with macrolide resistance. Significance and Impact of the StudyThis multiplex method can potentially simplify the identification of Camp.jejuni and determine macrolide resistance due to mutations in 23S rDNA or ribosomal protein L4. This method has a potential for application in different research areas and molecular surveillance.