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Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. <i>Lacticaseibacillus rhamnosus</i> X253 is a potential probiotic strain with antioxidant capacity. Comparative genomics and single nucleotide polymorphisms (SNPs) were used to identify a strain-specific locus within the <i>holA</i> gene for strain X253 that was distinct in 30 different <i>L. rhamnosus</i> strains. Using quantitative PCR, the primers and probe designed for the locus were able to distinguish <i>L. rhamnosus</i> X253 from the other 20 probiotic strains. The chosen locus remained stable over 19 generations. The sensitivity of the assay was 0.2 pg genomic DNA of <i>L. rhamnosus</i> X253, or 10³ cfu/mL bacteria of this strain. In terms of repeatability and reproducibility, relative standard deviations (RSD) were less than 1% and 3%, respectively. Additionally, this assay achieved accurate enumerations of <i>L. rhamnosus</i> X253 in spiked milk and complex powder samples. The strain-specific assay could be used for quality control and compliance assessment of dairy products.