A reverse transcriptase PCR (RT-PCR) was used for rapid determination of the hemagglutinin (HA) cleavage site sequence, a marker for the virulence potential of avian influenza viruses. When applied to specimens from chickens experimentally infected with either a virulent or an avirulent virus, RT-PCR uniformly detected the HA gene, even in specimens that were negative for virus by standard testing in eggs. This technique, combined with sequencing of the HA cleavage site, offers a rapid and sensitive way to assess the virulence potential of avian influenza viruses. Early detection of field isolates with virulence-associated structural motifs at the HA cleavage site would allow better control of influenza among large poultry populations.