BackgroundOutbreaks of highly pathogenic avian influenza (HPAI) on poultry farms and in wild birds worldwide persists despite intensified control measures. It causes unprecedented mortality in bird populations and is increasingly affecting mammalian species. Better understanding of HPAI introduction pathways into farms are needed for targeted disease prevention and control. The relevance of airborne transmission has been suggested but research involving air sampling is limited and unequivocal evidence on transmission routes is lacking.AimWe aimed to investigate whether HPAI virus from wild birds can enter poultry houses through air inlets by characterising host materials through eukaryote DNA sequencing.MethodsWe collected particulate matter samples in and around three HPAI-affected poultry farms which were cleared and decontaminated before sampling. Indoor measurements (n = 61) were taken directly in the airflow entering through air inlets, while outdoor air samples (n = 60) were collected around the poultry house. Positive controls were obtained from a bird rehabilitation shelter. We performed metabarcoding on environmental DNA by deep sequencing 18S rRNA gene amplicons.ResultsWe detected waterbird DNA in air inside all three, and outside of two, poultry farms. Sequences annotated at species level included swans and tufted ducks. Waterbird DNA was present in all indoor and outdoor air samples from the bird shelter.ConclusionAirborne matter derived from contaminated wild birds can potentially introduce HPAI virus to poultry houses through air inlets. The eDNA metabarcoding could assess breaches in biosecurity for HPAI virus and other pathogens potentially transmitted through air via detection of their hosts.