The diagnosis of avian influenza (AI) virus infections, even highly pathogenic AI (HPAI), represents a considerable challenge due to the lack of pathognomonic or specific clinical signs and their variation in different avian hosts plus the marked antigenic variation amongst influenza A viruses. Conventional laboratory techniques involve the isolation, identification and characterization (including virulence estimates) of the virus. While this has proven successful in the past and remains the method of choice, for at least the initial outbreak, the delays associated with conventional diagnosis are often considered unacceptable for the application of control measures, especially stamping out policies, and there is an overwhelming demand for rapid results. More and more, molecular biological techniques are being used and in particular reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR technologies are being employed for rapid diagnosis. In this paper, clinical signs, the molecular basis for virulence of AI viruses, international definitions, conventional diagnosis and the use of molecular techniques are reviewed and discussed.