This study aimed to evaluate the performance of polymerase chain reaction (PCR) methods used for the diagnosis of pertussis in laboratories within Europe in 2011. National reference laboratories in 25 European countries were contacted and a total of 24 laboratories from 19 countries agreed to participate in the study. A panel of seven samples of DNA from Bordetella pertussis, Bordetella parapertussis and Bordetella holmesii plus a negative control were distributed and analysed according to the routine PCR methods in each laboratory. The study took place in 2011. Nineteen laboratories used a real-time PCR approach, four laboratories used block-based PCR and one laboratory used a combination of methods. Six different combinations of amplification targets were used, and ten laboratories tested only for the presence of B. pertussis DNA. All laboratories (24/24) correctly identified a sample with high concentration of B. pertussis DNA, while three misidentified the B. parapertussis DNA as B. pertussis and 15 misidentified the B. holmesii DNA as either B. pertussis or B. parapertussis. There was a wide variation in the methods used for PCR-based diagnosis of pertussis among the European laboratories. Several laboratories were not able to discriminate between DNA samples from different Bordetella species.