Exact species/subspecies identification is essential in understanding human-associated microbiota and the practical application of a particular bacterial isolate. With the fast evolution of DNA sequencing technology, however, there is a transition in molecular identification methods from single gene sequence- to whole genome sequence analysis, resulting in subtle changes in bacterial taxonomy. Here we report the application of three different species identification molecular methods and species delineation concepts to identify a human breast milk isolate Bifidobacterium sp. 2450 - classical 16S-rDNA sequence analysis, multilocus sequence typing (MLST) and digital DNA:DNA hybridization (dDDH). Comparison of the partial 16S-rDNA sequence of Bifidobacterium sp. 2450 and type strains of the Bifidobacterium genus positioned the new isolate in the B. longum cluster. At subspecies level, including the four subspecies of B. longum (longum, infantis, suis and suillum), the partial 16S-rDNA sequence derived from Bifidobacterium sp. 2450 was >99.5% (1490 bp) identical to B. longum ssp. infantis. MLST was based on the concatenated partial sequences of the clpC, dnaG, dnaJ1, hsp60, purF, rpoC and xfp genes. Unlike16S-rDNA sequence analysis, MLST situated Bifidobacterium sp. 2450 closer to the B. longum ssp. longum cluster, separately from B. longum ssp. infantis. Next, dDDH was performed to compare the draft genome of Bifidobacterium sp. 2450 to complete genomes of type strains. Hybridization values with the type strain of B. longum ssp. longum DSM 20219 were 75.8 against only 62.4 for B. longum ssp. infantis DSM 20088. With a species delineation threshold for dDDH of 70, this method identified Bifidobacterium sp. 2450 as B. longum ssp. longum. Although the results from the three methods disagreed at intraspecies level, they all confirmed the new isolate to belong to the B. longum species, an identification level that is satisfactory for practical purposes.