A total of 3425 influenza B viruses collected from the Asia-Pacific region were tested against the four registered neuraminidase inhibitors (NAIs) (oseltamivir carboxylate, zanamivir, peramivir and laninamivir) as part of the routine surveillance work at the WHO Collaborating Centre for Research and Reference on Influenza, Melbourne between 2016 and 2020. Forty-five influenza B viruses with reduced susceptibility to one or more NAIs were identified. While the majority of these had neuraminidase (NA) mutations that were known to confer NAIs resistance, fifteen had NA mutations that had not been confirmed as being responsible for reduced NAIs susceptibility. Eleven of these NA mutations of concern were investigated using reverse genetics (RG) techniques to verify that these mutations were the cause of the reduced NAI susceptibility. All mutations were introduced separately into the NA of B/Brisbane/27/2016 (a B Victoria-lineage virus) or B/Yamanashi/166/98 (a B Yamagata-lineage virus) and the effects of these were analysed by an in vitro NAI assay. The T146K substitution in the NA of B Victoria and Yamagata-lineages resulted in a large increase in the IC50 for peramivir (>1000-fold increase in the mean IC50 of sensitive viruses with T146) with smaller increases for zanamivir and oseltamivir. A proline substitution (T146P) had a slightly lower (>700-fold) effect on the peramivir IC50 and also on the other NAIs. The presence of a second NA mutation at N169S combined with the T146P further increased the IC50 of peramivir (>7000-fold) and the other NAIs. A synergistic effect was also confirmed for dual NA mutations with G247D + I361V which showed a modest increase in the IC50 for oseltamivir (6-fold). Only one of two RG-viruses with the mutation G108E could be rescued and it had a high IC50 against zanamivir (>4000-fold) and laninamivir (>7000-fold), but a lower IC50 against oseltamivir (>200-fold). NA mutations H101L, A200T, D432G, H439P and H439R were also confirmed to somewhat reduce the in vitro susceptibility of influenza B viruses to the NAIs. Overall, this study identifies the potential impact of selected mutations on the clinical performance of NAIs when used to treat influenza B infection in humans.