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Current predominant routes of group B Streptococcus (GBS) transmission in preterm neonates admitted to neonatal intensive care unit (NICU) are poorly defined. We report 2 overlapping clusters of GBS late-onset disease (LOD) from June to September 2015 in an Italian NICU.During the outbreak, possible sources of transmission (equipment, feeding bottles and breast pumps) were swabbed. Specimens from throat and rectum were collected on a weekly basis from all neonates admitted to NICU. Colonized or infected neonates had cohorting. Bacterial isolates were characterized by serologic and molecular typing methods.GBS was isolated in 2 full-term and 7 preterm neonates. Strains belonged to serotype III, with 3 different sequence types (ST17, ST182 and ST19). Full-term neonates were colonized with GBS strains unrelated to the clusters (ST182 and ST19). Two distinct ST17 strains caused 2 clusters in preterm neonates: a first cluster with 1 case of LOD and a second, larger cluster with 6 LOD in 5 neonates (one of them had recurrence). ST17 strains were isolated from vaginorectal and milk samples of 2 mothers. Two preterm neonates had no evidence of colonization for weeks, until they presented with LOD.Molecular analyses identified the presence of multiclonal GBS strains and 2 clusters of 7 cases of GBS–LOD. The dynamics of transmission of GBS within the NICU were complex. Breast milk was suspected to be one of the possible sources. In a research setting, the screening of GBS carrier mothers who deliver very preterm could contribute to the tracking of GBS transmission.