Influenza A viruses, which are further subtyped on the basis of antigenic differences in external hemagglutinin and neuraminidase glycoproteins, and influenza B viruses are prominent among the viral causes of respiratory diseases and can cause a wide spectrum of illness. Each year these viruses are responsible for recurrent epidemics, frequently in association with genetic variation. There is a requirement for sensitive and rapid diagnostic techniques in order to improve both the diagnosis of infections and the quality of surveillance systems. A new three-dimensional biochip platform (Flow-Thru Chip; MetriGenix) was used to develop a rapid and reliable molecular method for the typing and subtyping of influenza viruses. Oligonucleotide probes immobilized in microchannels of a silicon wafer were selected to recognize multiple fragments of the influenza A virus matrix protein gene; the influenza B virus NS gene; the H1, H3, and H5 hemagglutinin genes; and the N1 and N2 neuraminidase genes. Biotinylated amplicons resulting from either multiplex or random reverse transcription-PCR were hybridized to arrayed oligonucleotides on the influenza virus chip before they were stained with horseradish peroxidase-streptavidin and were imaged by use of a chemiluminescent substrate. The chip analysis procedure, from the time of pipetting of the sample into the chip cartridge to the time of analysis of the results, was performed in less than 5 h. The random PCR exhibited a higher level of performance than the multiplex PCR in terms of the specificity of product hybridization to the influenza virus chip. Analysis of influenza A viruses (H1N1, H3N2, H1N2, and H5N1) and influenza B viruses showed that this microarray-based method is capable of the rapid and unambiguous identification of all types and subtypes of viruses by use of random PCR products. The redundancy of the probes designed for each gene selected yielded an additional criterion of confidence for the subtyping of viruses which are known for antigenic variations in some of their components.