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Single-nucleotide polymorphisms (SNPs) are the third generation of genetic markers, having been refined from the first generation of restriction fragment length polymorphisms and the second generation of microsatellite polymorphisms. SNPs represent a focal point of current studies of n Staphylococcus aureus. On one hand, this review aims to summarize common methodologies for detecting SNPs. These methods have typically included DNA genome sequencing methods and PCR-based detection methods. Alternative methods, such as mass spectrometry, denaturing high-performance liquid chromatography, SNaPshot, and SNP array have also been employed for SNP analysis. On the other hand, we enumerate a series of applications of SNP analysis in investigations of n Staphylococcus aureus. SNP analysis can be applied to investigate epidemiological outbreaks and transmission of n Staphylococcus aureus infections, the transmission and evolution of antimicrobial resistance genes in n Staphylococcus aureus isolates, interactions of n Staphylococcus aureus with other bacteria, and the links between n Staphylococcus aureus in humans and livestock.n “,”Single-nucleotide polymorphisms (SNPs) are the third generation of genetic markers, having been refined from the first generation of restriction fragment length polymorphisms and the second generation of microsatellite polymorphisms. SNPs represent a focal point of current studies of n Staphylococcus aureus. On one hand, this review aims to summarize common methodologies for detecting SNPs. These methods have typically included DNA genome sequencing methods and PCR-based detection methods. Alternative methods, such as mass spectrometry, denaturing high-performance liquid chromatography, SNaPshot, and SNP array have also been employed for SNP analysis. On the other hand, we enumerate a series of applications of SNP analysis in investigations of n Staphylococcus aureus. SNP analysis can be applied to investigate epidemiological outbreaks and transmission of n Staphylococcus aureus infections, the transmission and evolution of antimicrobial resistance genes in n Staphylococcus aureus isolates, interactions of n Staphylococcus aureus with other bacteria, and the links between n Staphylococcus aureus in humans and livestock.n