Abstract: The ability to rapidly detect wild polioviruses in clinical specimens is a major concern for the world-wide eradication of polioviruses. Provided is a method of detecting polioviruses of all three serotypes from viral isolates of clinical specimens using a pair of degenerate PCR primers. This primer set, which uses deoxyinosine residues to compensate for third position mismatches at specific positions, recognizes nucleotide sequences near the receptor binding site of polioviruses. These sequences are unique to polioviruses and are absolutely conserved at the amino acid level. As a result, these PCR primers do not recognize nonpoliovirus enteroviruses. All poliovirus serotypes (40 poliovaccine related genotypes and 120 wild poliovirus genotypes from around the world) tested positive. All 14 prototype strains of nonpoliovirus enteroviruses tested negative.