A single nucleotide polymorphism (SNP) was detected by the use of affinity chromatography with a single-stranded DNA ligand and temperature gradient elution. Because of the difference in thermostability between the ligand and sample single-stranded DNAs with and without SNPs, they were eluted as a peak with a shoulder by the temperature gradient elution. The sample DNAs with SNPs were detected as low as 0.1 pmol, and the elution behavior of DNAs with and without SNPs in this chromatography was predicted by a numerical calculation method based on a thermodynamic mass transfer model.