We retrospectively investigated mother-to-infant transmission of group B Streptococcus (GBS) in 98 cases of late-onset disease reported during 2007–2018 by a network in Italy. Mothers with full assessment of vaginal/rectal carriage tested at prenatal screening and at time of late onset (ATLO) were included. Thirty-three mothers (33.7%) were never GBS colonized; 65 (66.3%) were vaginal/rectal colonized, of which 36 (36.7%) were persistently colonized. Mothers with vaginal/rectal colonization ATLO had high rates of GBS bacteriuria (33.9%) and positive breast milk culture (27.5%). GBS strains from mother–infant pairs were serotype III and possessed the surface protein antigen Rib. All but 1 strain belonged to clonal complex 17. GBS strains from 4 mother–infant pairs were indistinguishable through pulsed-field gel electrophoresis. At least two thirds of late-onset cases are transmitted from mothers, who often have vaginal/rectal carriage, positive breast milk culture, or GBS bacteriuria, which suggests heavy maternal colonization.