A rapid duplex real-time polymerase chain reaction (PCR) assay for speciation of Campylobacter jejuni and Campylobacter coli using the ABI Prism 7700 sequence detection system (Applied Biosystems) was developed based on two of the genes used in a conventional multiplex PCR. A rapid turnaround time of 3 h was achieved with the use of boiled cell lysates. Applicability of the assay was tested with 6015 random campylobacter strains referred to the Campylobacter Reference Unit, with 97.6% being identified as either C. jejuni or C. coli by this technique. Rapidity, combined with specificity and sensitivity, makes this method for routine campylobacter speciation attractive to any laboratory with a Taqman system.