BACKGROUND Haemophilus influenzae (Hi) serotype b (Hib) conjugate vaccine was incorporated into the infant immunisation schedule in Brazil in 1999, where Hib was one of the major etiologic sources of community-acquired bacterial meningitis. OBJECTIVES The purpose of this study is to describe the molecular epidemiology of invasive Hi disease in Rio de Janeiro state, Brazil, before and after vaccine introduction. METHODS Surveillance data from 1986 to 2014 were analysed. Hi isolates recovered from cerebrospinal fluid (CSF) or blood from 1993 to 2014 were serotyped by slide agglutination, genotyped by multilocus sequence typing (MLST), and the capsule type evaluation, differentiation of serologically non-typeable isolates, and characterisation of the capsule (cap) locus was done by polymerase chain reaction. Antimicrobial susceptibility testing was performed using E-test. FINDINGS From 1986 to 1999 and from 2000 to 2014, 2580 and 197 (42% without serotype information) confirmed cases were reported, respectively. The case fatality rate was 17% and did not correlate with the strain. Hib and b-variant isolates belonged to ST-6, whereas serotype a isolates belonged to the ST-23 clonal complex. Serotype a appeared to emerge during the 2000s. Non-encapsulated isolates were non-clonal and distinct from the encapsulated isolates. Ampicillin-resistant isolates were neither of serotype b or were non-encapsulated, and all of them were β-lactamase-positive but amoxicillin-clavulanic acid susceptible. MAIN CONCLUSIONS Although Hi meningitis became a relatively rare disease in Rio de Janeiro after the introduction of the Hib conjugate vaccine, the isolates recovered from patients have become more diverse. These results indicate the need to implement an enhanced surveillance system to continue monitoring the impact of the Hib conjugate vaccine.