Insertion sequences IS481 and IS1001 are targets for molecular detection of respectively Bordetella pertussis and Bordetella parapertussis. There is a raising concern about specificity of these targets due to sequence similarity with Bordetella holmesii and Bordetella bronchiseptica. The likelihood of false (para)pertussis diagnoses should be correlated with the prevalence of these organisms in the respiratory tract (RT). From October 2010 until September 2011, 2,207 RT samples were submitted to the Belgian reference laboratory for pertussis diagnosis. End-point IS481/IS 1001 PCR and culture were performed for B. pertussis and B. parapertussis. We developed a sensitive culture method followed by screening with matrix-assisted laser desorption/ionisation- time of flight mass spectrometry (MALDI-TOF MS) to look for B. holmesii and B. bronchiseptica in our samples,. Only one B. bronchiseptica and no B. holmesii were detected in RT samples from Belgian patients with pertussis-like symptoms.