Using clinical materials from experimentally infected poultry, we established an effective method for the preparation of viral RNA directly from tissue samples and eggs. Furthermore, our type A–specific matrix reverse transcription–polymerase chain reaction (RT-PCR) test was improved, and an H7 subtype–specific nested RT-PCR, which includes the hemagglutinin cleavage site, was designed. Both RT-PCR systems proved to be as sensitive as virus isolation. In addition, the labeled H7 HA-nested PCR primers were suitable for sequencing of the PCR products. The RT-PCR amplification of viral RNA and sequencing of the PCR product allows for the sensitive and rapid differentiation between low-pathogenic and highly pathogenic avian influenza viruses.