A triplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single-target real-time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony-forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra- and inter-assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture-positive samples were also PCR positive. Our single-tube triplex real-time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.