Breast milk is important for infant health. Some of its benefits are due to the presence of a specific population of bacteria in the microflora. However, the microbiome of breast milk is influenced by many parameters such as maternal diet, breastfeeding and geographic location. Culture and non-culture methods have been used in studies of this bacterial population worldwide. But in the DR Congo, there was no study reporting the use of culture-independent techniques to characterize the bacterial diversity of human milk. The aim of this study was to identify the bacterial 16S rRNA gene from two genera Lactobacillus and Bifidobacterium. The 16S rRNA gene was also identified from four species: Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium longum and Bifidobacterium lactis. This analytical cross-sectional study was conducted in Kinshasa. Breast milk from some healthy women was collected from February 2 to 28, 2018. A culture-independent protocol using the classical polymerase chain reaction (PCR) was used to identify the bacterial 16S rRNA gene. The 68 samples of breast milk were collected in a sterile condition. The bacteria-specific ribosomal gene 16S rRNA was detected in 91.18% of Lactobacillus and 32.35% of Bifidobacterium at genus level. At of species level, only Lactobacillus reuteri 16S rRNA gene was identified in 89.71%. The 16S rRNA gene from the other species could not be amplified. There was also an association between educational level and the presence of Bifidobacterium How to cite this paper: Nzingula, O.,