Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease, to severe neurological complications. Instead of switching genotypes seen in EV71 outbreaks during 1998-2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012. To examine whether EV71 strains from both genotype B5 outbreaks were the same, we analyzed the full-length sequences of isolates from Taiwan. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions in non-structural protein coding region predominantly; interestingly, seven were in 3D polymerase region. We compared B5 viruses of each from 2008 and 2012, and found 2012-EV71 had higher growth kinetics. We then constructed reverse genetics (rg) viruses containing 3D-2008 and 3D-2012-protein region, respectively, by using N7008TW99 (genotype B4) as the backbone. 3D-2012-rg virus showed faster replication rate than 3D-2008-rg virus, which was similar to native viruses. These findings suggest that the 3D protein region important for viral polymerase activity and replication play a role in displaying differential properties between 2008 and 2012 strains. The results indicate re-emergent genotype B5 in 2012 belonged to a new sub-lineage of B5 with higher replication than 2008 B5 strains, which may contribute to the large EV71 outbreak in 2012 in Taiwan.