In the Netherlands, influenza specific antivirals are used for the therapy of influenza in nursing homes and hospitals, for prophylaxis in high risk groups and neuraminidase inhibitors are stockpiled as part of pandemic preparedness plans. To monitor the antiviral susceptibility profile, human influenza virus isolates derived from the Dutch influenza surveillance in 2005-2006 (n = 87), 2006-2007 (n = 58) and 2007-2008 (n = 128) were analyzed with phenotypic assays and sequencing. For adamantanes, a high proportion (>74%) of A(H3N2) viruses had the S31N mutation in M2 protein, while variation in the HA1 region of adamantane-sensitive viruses suggested that adamantane-sensitive variants were reseeded into the Dutch population and re-emerged as drug-sensitive due to M-segment reassortment. For neuraminidase inhibitors oseltamivir and zanamivir, 98% of types A and B influenza viruses prior to 2007-2008 were sensitive for both, whereas 24% of the A(H1N1) viruses obtained in 2007-2008 were oseltamivir-resistant while retaining sensitivity to zanamivir and adamantanes. Furthermore, oseltamivir-resistant A(H1N1) or adamantane-resistant A(H3N2) virus infections were not associated with differences in clinical symptoms compared to infections with sensitive variants. Our data show the dynamic nature of emergence of drug-resistant influenza viruses, stressing the need for surveillance of resistance trends as part of influenza monitoring programs.