Lactobacillus represents one of the most important genera of human and animal intestinal tract. They are used in dairy and nondairy probiotics foods to restore the intestinal microflora, which provides a health benefit. Lactobacillus strains were phenotypically identified and described using biochemical features and kits such as the API 50 CH system. Conventional biochemical and physiological studies have certain limitations when discriminating against large numbers of isolates with similar physiological characteristics. They are generally unreliable, and consuming money, time, and effort. There are many studies have focused on the rapid identification of molecular biology techniques for the rapid identification and detection of lactobacilli. In this study, we examined five pairs of primers on complex communities to facilitate the examination of complex micro-ecosystems. The two primers Lac1-Lac2 and Joh16SI-Joh16II showed positive results with L. casei, L. rhamnosus, and L. gasseri. Their strains were isolated from raw milk and feces of breast-fed infants. The fragments obtained from DNA samples after PCR amplification were sequenced and submitted in GenBank "NCBI" under accessions number "KY123805", "KY123789" and "KY123806". As the long-term goal of this research was to obtain patent protection for Lactobacillus sp. isolated from Egyptian resources and using it in food and dairy industries.