BACKGROUND Haemophilus influenzae is the etiological agent of acute- (meningitis, sepsis, pneumonia, epiglottis) and chronic infections (cystic fibrosis, chronic obstructive pulmonary infections). Several clinical (chronic infections) and epidemiological situations (community and hospital outbreaks) require the use of epidemiological typing methods. OBJECTIVE We evaluated four typing methods to characterize clinical isolates of H. influenzae. METHODS Forty-five clinical strains of H. influenzae were studied by biotype, serotype and antibiotype techniques, and the following methods: isoenzyme electrophoresis mobility, outer membrane proteins (OMP), ribotyping and pulsed field gel electrophoresis (PFGE). RESULTS Type b strains had one pattern with Isoenzyme, OMP and ribotyping techniques. Only PFGE enabled us to differentiate between several type b patterns, although all of them were close related. All methods showed a great variety of patterns with the non-typable H. influenzae although clinical- or epidemiological-related strains had identical patterns. CONCLUSIONS Results obtained with all four markers studied showed good agreement. Type b strains, in contrast to non-typable strains, had a strong clonal structure with all the assayed techniques; only PFGE showed differences. In terms of technical and financial cost, as well as reproducibility and discriminative power, we suggest as a markers for H. influenzae OMP and PFGE.