A quantitative detection of mutations mediating high-level macrolide resistance in Campylobacter is necessary for molecular epidemiological investigation and research. In this study, a new TaqMan probe-based real-time polymerase chain reaction method, with high specificity and sensitivity, was firstly established to identify and quantify mutated alleles associated with macrolide resistance (A2074C and A2075G) in 23S rDNA of Campylobacter. Three standard plasmids (plasmid wild type, plasmid containing A2074C, and plasmid containing A2075G) were constructed to evaluate the specificity of the established TaqMan real-time polymerase chain reaction and to generate standard curve for absolute quantification. This assay, using specific probes, can particularly identify the three standard plasmids corresponding to three genotypes (wild type, A2074C tranversion, and A2075G transition). The linear detection range of mutated alleles was found between 10 and 10(6) copies with the correlation coefficient of 0.99. Twelve laboratory-induced macrolide-resistant derivations and 18 isolated Campylobacter strains were also tested to evaluate the accuracy of the new assay. The specificity and accuracy of our established method were 100% identical to DNA sequencing. The entire procedure of the assay takes less than 2 h, eliminating the need for gel electrophoresis. Compared with previously reported qualitative methods, this quantitative genetic assay is a rapid and sensitive and accurate method for analysis of mutated alleles in 23S rDNA associated with high-level macrolide resistance in Campylobacter.