| Summary: | <i>Bifidobacterium longum</i> is one of the most widely distributed and abundant <i>Bifidobacterium</i> in the human intestine, and has been proven to have a variety of physiological functions. In this study, 80 strains of <i>B. longum</i> isolated from human subjects were classified into subspecies by ANI and phylogenetic analyses, and the functional genes were compared. The results showed that there were significant differences in carbohydrate metabolism between the two subspecies, which determined their preference for human milk oligosaccharides or plant-derived carbohydrates. The predicted exopolysaccharide (EPS) gene clusters had large variability within species but without difference at the subspecies level. Four subtype CRISPR-Cas systems presented in <i>B. longum</i>, while the subtypes I-U and II-C only existed in <i>B. longum</i> subsp. <i>longum</i>. The bacteriocin operons in <i>B. longum</i> subsp. <i>infantis</i> were more widely distributed compared with <i>B. longum</i> subsp. <i>longum</i>. In conclusion, this study revealed the similarities and differences between <i>B. longum</i> subsp. <i>infantis</i> and <i>B. longum</i> subsp. <i>longum,</i> which could provide a theoretical basis for further exploring the probiotic characteristics of <i>B. longum</i>.
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