Virus isolation (VI) in specific-pathogen-free (SPF) embryonated eggs or cell cultures is traditionally considered the method of choice for the detection and identification of avian influenza (AI) viruses. However, its value is limited because it is time-consuming and not cost-effective. AI is a highly contagious disease, able to spread in a susceptible population in a short period of time. Therefore, the prompt identification of an infected flock is crucial for control and eradication purposes. During an AI outbreak, the sample processing times using the above mentioned methods are often not compatible with the demands of the poultry industry. In addition, the delay in moving birds from a premises, whilst awaiting the VI result, often results in animal welfare issues. For this reason, when dealing with an epidemic of AI, rapid and reliable laboratory tests, such as RT-PCR and real-time PCR, should be available to reveal direct evidence of infection in the flocks. Scientific reports have been published in the recent past, evaluating their use during AI monitoring and surveillance programmes and epidemics. Based on the available information, the advantages and limits concerning the application of molecular methods during AI outbreaks are discussed in this paper.