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<i>Bifidobacterium longum</i> subsp. <i>infantis</i> (<i>B. infantis</i>) is one of a few microorganisms capable of metabolizing human breast milk and is a pioneer colonizer in the guts of breastfed infants. One current challenge is differentiating <i>B. infantis</i> from its close relatives, <i>B. longum</i> and <i>B. suis</i>. All three organisms are classified in the same species group but only <i>B. infantis</i> can metabolize human milk oligosaccharides (HMOs). We compared HMO-metabolizing genes across different <i>Bifidobacterium</i> genomes and developed <i>B. infantis</i>-specific primers to determine if the genes alone or the primers can be used to quickly characterize <i>B. infantis</i>. We showed that <i>B. infantis</i> is uniquely identified by the presence of five HMO-metabolizing gene clusters, tested for its prevalence in infant gut metagenomes, and validated the results using the <i>B. infantis</i>-specific primers. We observed that only 15 of 203 (7.4%) children under 2 years old from a cohort of US children harbored <i>B. infantis</i>. These results highlight the importance of developing and improving approaches to identify <i>B. infantis</i>. A more accurate characterization may provide insights into regional differences of <i>B. infantis</i> prevalence in infant gut microbiota.