Objective To identify three Bifidobacterium strains using different methods and analysis of acquired antimicrobial resistance determinants and their transferability combined with antimicrobial susceptibility testing results and whole-genome sequencing (WGS) data.Methods Three Bifidobacterium strains were isolated from three probiotic products and identified using biochemical tests, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and WGS. The antimicrobial susceptibility of the strains to eight antibiotics, including ampicillin, was tested using the broth microdilution method. The WGS data were used to analyze the acquired antimicrobial resistance determinants carried by the strains and assess their transferability using online databases.Results The results showed that biochemical methods could provide reliable genus-level identification, MALDI-TOF MS could provide reliable species-level identification, and WGS-based ANI and DDH values could provide reliable species and subspecies-level identification. Combined with wgSNP and wgMLST analysis, all three strains were identified as Bifidobacterium animalis subsp. lactis. The three strains showed decreased susceptibility to tetracycline and gentamicin. The decreased susceptibility to gentamicin was associated with the lack of transport system in the anaerobes and was an intrinsic phenotype. All three strains carried tet(W) on their chromosomes, and there were IS5 sequences downstream of tet(W), suggesting that this resistance gene may be a mobile element. A comparison of tet(W) sequences from 58 bacteria of different species and genera in GenBank showed that tet(W) sequences in B. animalis subsp. lactis have a certain degree of conservation.Conclusion The identification and safety evaluation of Bifidobacterium should be comprehensively carried out by combining biochemical tests, MALDI-TOF MS, antimicrobial susceptibility, and WGS. WGS technology can not only perform precise identification of Bifidobacterium at the subspecies level but also conduct in-depth analysis of resistance determinants and transferability. It provides strong technical support for the research and development, supervision, and healthy development of related probiotic products and the food industry.