Bordetella holmesii is a bacterium recently recognized in 1995. It is a gram-negative coccobacillus that can cause pertussis-like symptoms in humans as well as invasive infections. It is often confused with Bordetella pertussis because routine diagnostic tests for whooping cough are not species-specific. The prevalence of B. holmesii as a cause of pertussis has increased in several countries. Therefore, B. holmesii assays are important for determining the epidemiology of pertussis, for the choice of an effective treatment, and for detecting vaccination failures.