This study aimed to isolate and genetically investigate the l. acidophilus. We collected two hundred milk product samples (130 from Assiut and 70 from New Valley) from different sources including yogurt (90), rayeb (80), and milk powder (30). Lactobacillus are isolated on MRS agar and the resulting colonies were purified by repeated subculture and preliminarily identified as Lactobacillus sp. based on Gram staining, morphology and biochemical tests. Complementary studies on aspects like the ability to grow at different temperatures and resistance to inhibitory substances such as bile, NaCl 4% and 5.5% were performed. Then, Genotypic investigation for isolation of L. acidophilus. The occurrence of lactobacillus in New Valley was 22.85% and 25.71% of yogurt and rayeb respectively and the occurrence of Lactobacillus in Assiut was 18.46%,16.15% and 6.92% of yogurt, rayeb and Milk powder respectively. After the genotypic characterization of these isolates, nineteen L. acidophilus have been isolated out of all samples under study.