Aims: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. Methods and Results: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. Conclusions: The bacterial composition of breast milk and infant faeces is not related to the delivery method. Significance and Impact of the Study: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.