Introduction Highly pathogenic avian influenza (HPAI) viruses of subtype H5N8 were re-introduced into the Netherlands by late 2016, after detections in south-east Asia and Russia. This second H5N8 wave resulted in a large number of outbreaks in poultry farms and the deaths of large numbers of wild birds in multiple European countries. Methods: Here we report on the detection of HPAI H5N8 virus in 57 wild birds of 12 species sampled during active (32/5,167) and passive (25/36) surveillance activities, i.e. in healthy and dead animals respectively, in the Netherlands between 8 November 2016 and 31 March 2017. Moreover, we further investigate the experimental approach of wild bird serology as a contributing tool in HPAI outbreak investigations. Results: In contrast to the first H5N8 wave, local virus amplification with associated wild bird mortality has occurred in the Netherlands in 2016/17, with evidence for occasional gene exchange with low pathogenic avian influenza (LPAI) viruses. Discussion: These apparent differences between outbreaks and the continuing detections of HPAI viruses in Europe are a cause of concern. With the current circulation of zoonotic HPAI and LPAI virus strains in Asia, increased understanding of the drivers responsible for the global spread of Asian poultry viruses via wild birds is needed.