Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) previously found in human infections into the NA of three current AIV subtypes of the H5Nx genotype that threaten the poultry industry and human health: highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we showed that a R292K substitution in H5N6 and H5N2 viruses significantly reduced susceptibility to three licenced NAIs: oseltamivir, zanamivir and peramivir. In contrast the mutations E119V, H274Y and N294S had more variable effects with NAI susceptibility being drug- and strain-specific. We measured the replicative fitness of NAI resistant H5N6 viruses and found that they replicated to comparable or significantly higher titres in primary chicken cells and in embryonated hens’ eggs as compared to wild type – despite the NA activity of the viral neuraminidase proteins being reduced. The R292K and N294S drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA): Y98F and A189T, respectively (H3 numbering) which reduced receptor binding properties possibly balancing the reduced NA activity seen. Our results demonstrate that the H5Nx viruses can support drug resistance mutations that confer reduced susceptibility to licenced NAIs and that these H5N6 viruses did not show diminished replicative fitness in avian cell cultures. Our results support the requirement for on-going surveillance of these strains in bird populations to include motifs associated with human drug resistance.Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.