Infectious clone technology provides an opportunity to study the molecular basis of arthropod-virus interactions in detail. This study describes the development of an infectious clone of the prototype yellow fever virus Asibi strain (YFV-As) with the purpose of identifying sequences or domains that influence infection dynamics in the mosquito vector. The full-length cDNA of YFV-As virus was produced from RT-PCR products of parental viral RNA. These were cloned into a low-copy-number plasmid previously used to develop the YFV-17D infectious clone (pACNR/FLYF-17D). Virus recovered from the infectious clone exhibited biological characteristics similar to those of the parental YFV-As, including replication kinetics, reactivity to flavivirus cross-reactive and YFV-specific antibodies and infection and dissemination rates in Aedes aegypti, the principal mosquito vector of YFV. These data provide the basis for future studies with chimeric Asibi/17D viruses to identify the determinants of vaccine attenuation in the vector.