Within mosquitoes, arboviruses encounter barriers to infection and dissemination that are critical determinants of vector competence. The molecular mechanisms responsible for these barriers have yet to be elucidated. The prototype Sindbis (SIN) strain, AR339, and viruses derived from this strain, such as TR339 virus, have limited infection and transmission potential in the medically important arthropod vector, Aedes aegypti (L.). However, the Malaysian SIN virus strain, MRE16, disseminates in nearly 100% of Ae. aegypti 14 d after oral infection. Here, we compare the spatial and temporal infection patterns of MRE16 and TR339 viruses in Ae. aegypti. The results indicate that a midgut escape barrier is primarily responsible for the significantly lower dissemination and transmission potentials observed after oral infection with TR339 virus. MRE16 and TR339 viruses now represent a well-characterized model system for the further study of virus determinants of vector infection, particularly determinants affecting the midgut escape barrier in Ae. aegypti.