Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future.Highly pathogenic avian influenza (HPAI) viruses constitute a continuous concern from public health, veterinary, and wildlife perspectives. Whereas aquatic wild birds serve as the main reservoir for low-pathogenic avian influenza (LPAI) viruses, the emergence of HPAI viruses is primarily the result of largescale poultry husbandry (1,16,23,39). Outbreaks of HPAI predominantly occur in poultry and are restricted to influenza A viruses of the H5 and H7 subtypes. The last decade has seen a marked increase in outbreaks of HPAI in poultry around the world. While most HPAI outbreaks have been controlled relatively quickly, the H5N1 HPAI virus has continuously been circulating in poultry since 1997 (7, 10). The H5N1 HPAI virus is also unusual in the unprecedented scale and geographical spread of the outbreak that it has caused; its transmission to a wide variety of mammalian species, including humans; and the introductions of H5N1 HPAI virus in wild birds (5,22,28,40). These recent introductions of H5N1 HPAI virus in wild birds and the subsequent spread of the virus throughout Asia, the Middle East, Africa, and Europe have put a focus on the role of wild birds in the geographical spread of the H5N1 HPAI virus (29). Large-scale surveillance programs have been implemented in several parts of the world to determine the ...