Aim: This study investigated the cultural method and 16S rRNA gene analysis to reveal the composition and diversity of lactic acid bacteria (LAB) from dairy origin (spontaneous fermented cow milk) in Nigeria. Methods: Six dairy samples which includes two raw cow milk, two raw goat milk and two fermented cow milk (nono) were collected and subjected to standard microbiological investigation using both cultural and molecular methods. The dairy samples were cultured on MRS media, and the isolates were identified using physiological and biochemical parameters. DNA of four selected probiotic LAB isolates from nono were amplified using PCR while the amplicons were electrophoresed in agarose gel, pre-stained with ethidium bromide and characterized by 16S rRNA gene analysis. The result of the DNA sequencing were analyzed using NCBI BLAST. Results: A total of 55 presumptive LAB were isolated. Twenty nine (29) Lactobacillus plantarum representing 52.7%, Pediococcus acidilactici 15(27.2%), Lactobacillus casei and Lactobacillus brevis 4(7.3%) while Lactobacillus fermentum 3(5.5%) were recorded respectively. Result of the gel electrophoresis revealed DNA size of approximately 1500bp. The selected probiotic LAB from nono used in this study were confirmed as Lactobacillus plantarum N17, Lactobacillus plantarum N24, Lactobacillus brevis N10 and Lactobacillus casei N1 based on 16S rRNA gene sequence analysis while the phylogenetic analysis revealed a 98-100% similarity with a high homology level which affirms the strain of the organism. Conclusion: This study has demonstrated the diversity of LAB existing in dairy samples known as fermented cow milk (nono) which could be harnessed as valuable sources for LAB isolation and potential probiotic organisms.