Enterovirus-A71 (EV-A71) is a common cause of hand-foot-and-mouth disease (HFMD) and, rarely, causes severe neurological disease. This study aimed to elucidate the epidemiological and genetic characteristics and virulence of EV-A71 strains isolated from children diagnosed with HFMD. Rectal and throat swabs were collected from 488 children with HFMD in Hanoi, Vietnam, in 2015-2016. From 391 EV-positive patients, 15 EVs, including coxsackievirus A6 (CV-A6; 47.1%) and EV-A71 (32.5%, n = 127), were identified. Of the 127 EV-A71 strains, 117 (92.1%) were the B5 subgenotype and 10 (7.9%) were the C4 subgenotype. A whole-genome analysis of EV-A71 strains showed that seven of the eight C4a strains isolated in 2016 formed a new lineage, including two possible recombinants between EV-A71 C4 and CV-A8. The proportion of inpatients among C4-infected children was higher than among B5-infected children (80.0% vs. 27.4%; P = 0.002). The virulence of EV-A71 strains was examined in human scavenger receptor class B2 (hSCARB2)-transgenic mice, and EV-A71 C4 strains exhibited higher mortality than B5 strains (80.0% vs. 30.0%, P = 0.0001). Thus, a new EV-A71 C4a-lineage, including two possible recombinants between EV-A71 C4 and CV-A8, appeared in 2016 in Vietnam. The EV-A71 C4 subgenotype may be more virulent than the B5 subgenotype.