Surveillance after introduction of Haemophilus influenzae serotype b vaccination in Spain identified 26 H. influenzae serotype e (HiE) isolates. Of these, 16 (61.5%) were recovered from patients aged >16 years and 10 (38.5%) from children <16 years of age. HiE caused respiratory infections in 14 patients (9 with pneumonia), conjunctivitis in 4, vaginitis in 2, abscess in 2, and cellulitis, peritoneal infection, sepsis and meningitis in 1 patient each. HiE was strongly clonal and highly resistant to ampicillin and cotrimoxazole, and the incidence of HiE infection did not increase over time.