Background.?During 2010-2011, influenza B viruses with a novel neuraminidase substitution, denoted I221V, (B/I221V) associated with reduced in vitro oseltamivir susceptibility were detected in North Carolina (NC).Methods.?We determined the prevalence of I221V among B viruses submitted to CDC for antiviral resistance surveillance, including all B viruses submitted to NC and South Carolina (SC) state laboratories, during October 2010-September 2011.We conducted chart reviews and phone interviews to characterize NC and SC patients with B/I221V versus wild-type B virus infection (B/WT).Results.?We detected I221V in 45 (22%) of 209 B viruses from NC and 8 (10%) of 82 B viruses from SC. We detected I221V in three (0.3%) of 881 B viruses tested from 45 other states. B/I221V infection was not associated with differences in underlying conditions or illness severity compared with B/WT infection. No patients with B/I221V infection received oseltamivir prior to specimen collection. Among patients who completed oseltamivir, those with B/I221V infection reported a longer duration until illness resolution (5 versus 3 days); P=0.02.Conclusions.?B/I221V co-circulated with B/WT in NC and SC during 2010-2011. I221V did not alter illness severity but may have reduced oseltamivir effectiveness. Thus, global surveillance for I221V is important.