Strains of Bifidobacterium longum subsp. infantis (B. infantis) are amongst the first to colonize the infant gut, partly due to their capacity to metabolize complex human milk oligosaccharides (HMO), and are proposed to play a key role in the development of the infant gut. Since early life, B. infantis supplementation is of high interest, and detailed phylogenetic, functional and safety characterization of the selected strains should be pursued. Using a combination of long and short-read sequencing technologies, we first decipher the genetic distance between different isolates of the same B. infantis strain. Using the same approach, we show that several publicly available genomes recapitulate this strain-level distance as compared to two of the first strains obtained in the 1950s. Furthermore, we demonstrate that the two 1950s B. infantis strains display different functional and safety attributes, as ATCC 15697 is resistant to streptomycin and shows a preference towards lacto-N-tetraose LNT and sialylated HMOs, while LMG 11588 is sensitive to all tested antibiotics and shows a preference towards fucosylated HMOs. Overall, our work highlights that the current diversity observed in B. infantis is likely underestimated and that strain selection within this subspecies must be the subject of scientific pursuit and associated evaluation.