Muller’s Ratchet is the process by which asexual populations lose fitness when subjected to repeated genetic bottlenecks(1). Similar bottlenecks may be common during transmission of arboviruses as they move between insect and vertebrate hosts. To determine the effect of Muller’s Ratchet on transmission of dengue viruses (DENV), progeny from 10 plaque purified populations of DENV derived from an infectious cDNA clone were subjected to repeated plaque-to-plaque passages. Although eight of the 10 clones were able to produce viable progeny after 10 plaque-to-plaque transfers, one no longer produced plaques after 3 and another after 4 passages and competition assays demonstrated that all 10 single plaque populations had lost fitness and plaque-to-plaque transfers lead to further fitness lose. Analyses of the envelope (E) genes of these populations found that the genetic diversity of the single plaque virus was reduced by more than ninety per cent compared to the parental population from which they were derived and that the repeated plaque-to-plaque transfer also led to a change in the consensus sequence of the populations. These results demonstrate that Muller’s Ratchet operates on populations of dengue virus during repeated bottleneck passages and that it may lead to their extinction.