We report the use of ResPlex III for genotyping influenza A viruses. The performance characteristics of the assay with regard to H5N1 are further evaluated. The ResPlex system incorporates a novel multiplex PCR technology, target-enriched multiplex PCR, to simultaneously amplify multiple molecular targets in one reaction. The ResPlex III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the influenza A virus as well as the NS genes from influenza A (NSA) and B (NSB) viruses, providing detection and genotyping of influenza A and B viruses. The analytical sensitivities for detecting the H5, N1, and NSA genes were 1, 10(-1), and 10 50% tissue culture infectious doses/200 microl/reaction, respectively. A total of 217 sequential clinical samples including 14 samples with human H5N1 infections were tested by the ResPlex III assay, and the results were compared to a reference standard combined with results of viral culture and conventional reverse transcriptase and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were 93.3% and 100%, respectively, indicating that different subtypes of influenza A virus can be quickly and correctly identified using the ResPlex III genotyping approach.