Haemophilus influenzae serotype b (Hib) was the leading cause of bacterial meningitis in children before the implementation of infant immunization with conjugate Hib vaccines. Despite the effectiveness of the vaccine, invasive Hib disease cases (i.e. isolation of Hib from a normally sterile site) are still reported in children. All invasive Hib disease cases in children ≤ 15 years reported through the National Surveillance System of Invasive Bacterial Disease, during 2012-2018 in Italy, were analyzed. Hib PCR-confirmed isolates were subjected to MLST and PFGE analysis. The number of copies of the capb locus, a virulence factor potentially contributing to true vaccine failures (TVFs), was determined by Southern blot analysis. Vaccine effectiveness (VE) was determined using a multiple Poisson regression model. 31 cases of invasive Hib disease in children were reported. Fourteen children were vaccinated (TVFs), 14 were unvaccinated and 2 partially vaccinated (vaccination status was unknown for 1 case). The median age of children was 12 months (range 3 months-15 years). A decrease in vaccination coverage was observed in 2014-2016 (source Ministry of Health), and a rise in incidence was documented from 2016 until 2018, especially in children < 5 years. Vaccine effectiveness was estimated to be 83% (95% CI:45-95). 24 isolates were available. The predominant ST was ST6 (70.8%). Cluster analysis of ST6 isolates by PFGE identified five variants. Six isolates (25%) contained multiple copies of the capb locus distributed among TVFs (30%) and unvaccinated children (16.7%). Our data show that both failures to vaccinate and TVFs are associated with invasive Hib disease in children in Italy, during the vaccination era. Most cases in children ≤ 2 years were vaccine-preventable, since they occurred in unvaccinated subjects (13/21 cases, 62%). No host predisposing factors for TVF were recognized. TVFs were not significantly associated with either specific genotypes or amplification status of the capb locus.Copyright © 2020 Elsevier Ltd. All rights reserved.