Neuraminidase inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs-particularly newly introduced treatments-is critical to manage viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (ie, D199E and P458T) were shown to confer resistance to more than one NAI. Of note, mutants possessing P458T-which is located outside of the catalytic or framework residue of the NA active site-exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower compared with WT. Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with WT. Reverse mutations to WT were observed in the lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified which could possibly emerge in influenza A(H1N1)pdm09 during laninamivir therapy and have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading.