A multiplex polymerase chain reaction (PCR) assay has been developed for the identification of the four thermophilic Campylobacter commonly associated with human gastroenteritis including C. jejuni, C. coli, C. lari and C. upsaliensis. The best combination of primers and the annealing temperature of multiplex PCR were examined. Detection limit was 2 × 105 cfu and 100 ng of DNA or whole-cell suspension. The multiplex PCR was applied for the direct detection and differentiation of Campylobacter species in 33 human and 45 chicken caeca isolates. Of the 78 specimens evaluated by the multiplex PCR, 55 (70.5%) were identified as C. jejuni, 18 (23.0%) as C. coli and 5 (6.41%) as a mixed infection with both species. Comparison of hippurate test and multiplex PCR demonstrated five (6.41%) isolates with false-positive hippurate enzymic activity and three (3.85%) with false-negative activity. cdtB gene was detected in 100% and 38.9% of C. jejuni and C. coli, respectively. This multiplex PCR was found to be rapid, easy to perform and had a high sensitivity and specificity, even with mixed cultures. The system is useful for the detection of the presence of cdtB gene that is responsible for toxin activity in Campylobacter. © 2009, Wiley Periodicals, Inc.