Forty-four previously characterized strains of Haemophilus influenzae were used to evaluate the specificity of previously published SNP PCR primers for the detection of the N526K substitution in PBP3 of BLNAR isolates using real-time PCR. Hasegawa et al. primers that amplify strains without a substitution at 526 and fail to amplify strains with N526K were 100% sensitive and specific for detecting N526K. However, primer sets of Hasegawa et al. and Nakamura et al. designed to amplify strains with N526K, but not strains without a substitution, were unable to do this reliably because the primers were specific for N526K encoded by AAG and failed to amplify strains with N526K encoded by AAA. A review of N526K strains deposited on GenBank revealed an even distribution of AAG and AAA codons for N526K in European and Australian BLNAR strains, whereas only the AAG codon was seen in Japanese strains. The exclusive presence of the AAG codon in Japanese strains appears to be independent of the use of the SNP PCR primers evaluated here and remains unexplained.