We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002-2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989-2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1%) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0%, p < 0.001). Serotype b strains decreased significantly (from 81.0 to 13.2%, p < 0.001) and serotype f increased significantly (from 0.8 to 6.9%, p = 0.03). Ten percent of the isolates were β-lactamase producers, a value lower than that previously observed (26.9%, p = 0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.