Two point mutations (A2074C and A2075G) in the 23S rRNA gene of Campylobacter coli and C. jejuni are associated with erythromycin resistance. A real-time fluorescence resonance energy transfer PCR assay using a melting curve analysis was developed to identify these two point mutations and the wild-type genotype in thermophilic Campylobacter species such as C. coli, C. jejuni, and C. lari. Concerning these species, 141 strains were tested and a result obtained in 140 (99.3%). A single genotype was detected in 133 cases and two genotypes in seven cases. There was an agreement with the phenotypic methods except for one C. coli strain that was not amplified during the PCR assay. The A2075G mutation was mainly found among the 92 resistant strains tested (95.6%) and the A2074C mutation only twice (2.2%). One C. jejuni strain (1.1%) harbored both mutations on the same 23S rDNA copy. When compared to the phenotypic tests, the new real-time PCR assay was able to detect the correct genotype, in all cases except one (1.1%). This assay is more sensitive and more rapid than other PCR assays, the entire procedure taking less than 2 hr.