When we placed a 2'-O,4'-C-ethylene nucleic acid (ENA) residue into primers at the 3' end, or the n-1, n-2 or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. When we compared n-2-position-modified ENA with the corresponding unmodified DNA primers that are often used for allele-specific PCR (AS-PCR), a greater discrimination of the SNP site by ENA primers was observed. This improvement is probably due to the difficulty of incorporating a nucleotide into a mismatched ENA primer by Taq DNA polymerase in the primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.