Lactobacillus is a fairly diverse genus of bacteria with more than 260 species and subspecies. Many profiling methods have been developed to carry out phylogenetic analysis of this complex and diverse genus, but limitations remain since there is still a lack of comprehensive and accurate analytical method to profile this genus at species level. To overcome these limitations, a Lactobacillus-specific primer set was developed targeting a hypervariable region in the groEL gene-a single-copy gene that has undergone rapid mutation and evolution. The results showed that this methodology could accurately perform taxonomic identification of Lactobacillus down to the species level. Its detection limit was as low as 104 colony-forming units (cfu)/mL for Lactobacillus species. The assessment of detection specificity using the LactobacillusgroEL profiling method found that Lactobacillus, Pediococcus, Weissella, and Leuconostoc genus could be distinguished, but non-Lactobacillus Genus Complex could not be detected. The groEL gene sequencing and Miseq high-throughput approach were adopted to estimate the richness and diversity of Lactobacillus species in different ecosystems. The method was tested using kurut (fermented yak milk) samples and fecal samples of human, rat, and mouse. The results indicated that Lactobacillus mucosae was the predominant gut Lactobacillus species among Chinese, and L. johnsonii accounted for the majority of lactobacilli in rat and mouse gut. Meanwhile, L. delbrueckii subsp. bulgaricus had the highest relative abundance of Lactobacillus in kurut. Thus, this groEL gene profiling method is expected to promote the application of Lactobacillus for industrial production and therapeutic purpose.