The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.