Amino acid changes in or near the active site of neuraminidase (NA) in influenza viruses reduce the susceptibility to NA inhibitor drugs. Here, we report the recovery of three influenza B viruses with reduced susceptibilities to NA inhibitors from human patients with no history of antiviral drug treatment. The three viruses were isolated by inoculating Madin-Darby canine kidney (MDCK) cells with respiratory specimens from the patients. NA inhibition assays demonstrated that two of the three isolates showed a highly reduced susceptibility to peramivir and moderately reduced susceptibility to oseltamivir, zanamivir, and laninamivir. The remaining one isolate exhibited moderately reduced sensitivity to peramivir, zanamivir, and laninamivir but was susceptible to oseltamivir. A sequence analysis of viruses propagated in MDCK cells revealed that all three isolates contained a single mutation (Q138R, P139S, or G140R) in NA not previously associated with reduced susceptibility to NA inhibitors. However, pyrosequencing analyses showed that the Q138R and G140R mutations were below a detectable level in the original clinical specimens; the P139S mutation was detected at a very low level, suggesting that the mutant viruses may be preferably selected during propagation in MDCK cells. The NA crystallographic structure showed that these mutations were located at the interface between the two monomers of the NA tetramer, away from the NA active site. In addition to amino acid substitutions around the active site of NA, these observations suggest that alterations in the monomer-monomer interface region of NA may contribute to reduced sensitivity to NA inhibitors.