In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.