The 4 dengue virus serotypes (DENV-1-4) cause the most prevalent mosquito-borne viral disease of humans worldwide. DENV-2 Asian 1 (A1) genotype viruses replaced the Asian-American (AA) genotype in Vietnam and Cambodia, after which A1 viruses containing Q or M at envelope (E) residue 160 became more prevalent than those with residue 160K in both countries (2008-2011). We investigated whether these substitutions conferred a fitness advantage by measuring neutralizing antibody titer against reporter virus particles (RVPs) representing AA, A1-160K, A1-160Q, and A1-160M, using patient sera from Vietnam and a well-characterized Nicaraguan cohort. Surprisingly, we found that A1-160Q and A1-160M RVPs were better neutralized by heterologous antisera than A1-160K. Despite this, Vietnamese patients infected with A1-160Q or A1-160M viruses had higher viremia levels than those infected with A1-160K. We thus found that independent lineages in Vietnam and Cambodia acquired a substitution in E that significantly increased polyclonal neutralization but nonetheless were successful in disseminating and infecting human hosts.