The incidence of whooping cough in Chile ranges from 4.1 and 7.5 per hundred thousand inhabitants. B. pertussis detection is performed by Real Time PCR (Q-PCR) directed to the insertion sequence IS481. However, this sequence is also found in the genome of B. bronchiseptica and B. holmesii. The latter is also a respiratory pathogen whose clinical features are similar to B. pertussis. However, it is important to differentiate between these species because in immunosuppressed patients B. holmesii is more likely to cause bacteremia and is less susceptible to erythromycin. The goal of this work is to measure prospectively and retrospectively the presence of B. holmesii in samples reported positive for B. pertussis in the period 2010-2011. During this period, 1994 nasopharyngeal specimens entered the laboratory for Bordetella sp. PCR, of which 224 were positive. The analysis by Q-PCR directed to the recA gene of B. holmesii of all 224 positive samples determined a prevalence of B. holmesii of 0.6% (12/1994). Because of its more aggressive behavior in immunosupressed patients and its different resistance pattern, routine screening of B. pertussis and B. holmesii is currently performed for all samples in which Bordetella sp PCR is initially detected.