Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of Bifidobacterium breve metabolize four distinct alpha-glucose- and/or alpha-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all B. breve strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of alpha-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of alpha-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.