In Japan, the increasing incidence of β-lactum-resistant Haemophilus influenzae infections is of growing concern. We retrospectively studied whether the prevalence of β-lactamase-negative ampicillin-resistant strains of H. influenzae was influenced by chronic lung diseases. H. influenzae isolates, obtained from patients who were diagnosed with acute or chronic bronchitis, or acute exacerbation of chronic bronchitis in 2005, were studied. In addition to susceptibility testing, polymerase chain reaction (PCR) was performed for the detection of TEM-1 β-lactamase, and Asn526-Lys and Ser385-Thr amino acid substitutions in the ftsI gene encoding penicillin-binding protein-3 (PBP-3). The minimum inhibitory concentration values of β-lactams were found to be increased in isolates from patients with chronic bronchitis who had been repeatedly administered antibiotics. Genetic analysis using PCR suggested that this might be associated with a high frequency of β-lactamase-negative strains with mutations in PBP-3. The presence of β-lactum-resistant strains needs to be considered for patients with chronic bronchitis in whom H. influenzae is isolated as a causative pathogen.