Background: Haemophilus influenzae, a major pathogen causing respiratory tract infections and meningitis, is becoming increasingly resistant to narrow spectrum penicillin. Investigating the reasons for this resistance is challenging. Methods: The sequences of the ftsI gene, encoding the transpeptidase domain of penicillin binding protein (PBP3), were determined for 44 strains of Haemophilus influenzae with reduced susceptibility to β-lactam antibiotics. Strains, isolated from children, were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). Results: Sequence analysis of the ftsI gene revealed different mutations. We used this polymorphism to classify the different strains into three groups: (I (n=3), II (n=36, including H. influenzae ATCC 49247), this group was divided into four subgroups and III (n=5)). In each group various substitutions were observed. Conclusion: The increasing number of Haemophilus influenzae strains with reduced susceptibility to β-lactam due to mutations in the ftsI gene, is becoming a serious health issue in Tunisia. The resistance to β-lactam was observed in both strains that produce β-lactamase and those that do not. The level of resistance is remarkably high in our country.