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Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii , one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products. [ABSTRACT FROM AUTHOR]