Influenza viruses of the N1 neuraminidase (NA) subtype affecting both animals and humans caused the 2009 pandemic. Anti-influenza virus NA inhibitors are crucial early in a pandemic, when specific influenza vaccines are unavailable. Thus, it is urgent to confirm the antiviral susceptibility of the avian viruses, a potential source of a pandemic virus. We evaluated the NA inhibitor susceptibilities of viruses of the N1 subtype isolated from wild waterbirds, swine, and humans. Most avian viruses were highly or moderately susceptible to oseltamivir (50% inhibitory concentration [IC(50)], <5.1 to 50 nM). Of 91 avian isolates, 7 (7.7%) had reduced susceptibility (IC(50), >50 nM) but were sensitive to the NA inhibitors zanamivir and peramivir. Oseltamivir susceptibility ranged more widely among the waterbird viruses (IC(50), 0.5 to 154.43 nM) than among swine and human viruses (IC(50), 0.33 to 2.56 nM). Swine viruses were sensitive to oseltamivir, compared to human seasonal H1N1 isolated before 2007 (mean IC(50), 1.4 nM). Avian viruses from 2007 to 2008 were sensitive to oseltamivir, in contrast to the emergence of resistant H1N1 in humans. Susceptibility remained high to moderate over time among influenza viruses. Sequence analysis of the outliers did not detect molecular markers of drug-resistance (e.g., H275Y NA mutation [N1 numbering]) but revealed mutations outside the NA active site. In particular, V267I, N307D, and V321I residue changes were found, and structural analyses suggest that these mutations distort hydrophobic pockets and affect residues in the NA active site. We determined that natural oseltamivir resistance among swine and wild waterbirds is rare. Minor naturally occurring variants in NA can affect antiviral susceptibility.