This study aimed to isolate and characterize a new potential probiotic Lactobacillus with the aid of a culture method and PCR amplification of the 16S rRNA gene. The study employed a selective medium to identify fifty distinct Lactobacillus bacteria from five dairy samples sourced from the Dehradun region in Uttarakhand. Since the biochemical tests on all samples were nearly equivalent, one of the top five samples was chosen to undergo 16s RNA sequencing. Genes encoding 16S rRNA were amplified from DNA extracted from microbial cultures using universal bacterium primers using PCR. To identify the distant bacteria, the 16S rRNA genes of one identical bacteria were sequenced and aligned, and data was deposited in Gen Bank for the distant bacteria