Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3′ end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3′ end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3′ base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3′ end to block removal of the critical 3′ mutation-specific base by polymerases containing 3′ → 5′ exonuclease “proofreading” activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.