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<i>Lacticaseibacillus zeae</i> strains, isolated from raw milk and fermented dairy products, are closely related to the <i>Lacticaseibacillus</i> species that has beneficial probiotic properties. However, it is difficult to distinguish those using conventional methods. In this study, a unique gene was revealed to differentiate <i>L. zeae</i> from other strains of the <i>Lacticaseibacillus</i> species and other species by pan-genome analysis, and a real-time PCR method was developed to rapidly and accurately detect the unique gene. The genome analysis of 141 genomes yielded an 17,978 pan-genome. Among them, 18 accessory genes were specifically present in five genomes of <i>L. zeae</i>. The glycosyltransferase family 8 was identified as a unique gene present only in <i>L. zeae</i> and not in 136 other genomes. A primer designed from the unique gene accurately distinguished <i>L. zeae</i> in pure and mixed DNA and successfully constructed the criterion for the quantified standard curve in real-time PCR. The real-time PCR method was applied to 61 strains containing other <i>Lacticaseibacillus</i> species and distinguished <i>L. zeae</i> with 100% accuracy. Also, the real-time PCR method was proven to be superior to the 16S rRNA gene method in the identification of <i>L. zeae</i>.