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Two inbred (isofemale) Aedes aegypti mosquito lines were derived that manifested a resistant or susceptible phenotype following ingestion of yellow fever virus; lack of virus movement from the midgut defined the resistant phenotype. Other flaviviruses, including dengue 1–4, Uganda S, and Zika, viruses behaved in a similar fashion in the two mosquito lines. Crosses between the two lines produced progeny that were of intermediate susceptibility, indicating codominance; F2 backcrosses to the parents yielded results consistent with a major controlling genetic locus and provide evidence of a second locus capable of modulating the phenotype of the major gene. The rapid selection necessary to fix the susceptible and refractory phenotypes support the hypothesis of a single major controlling locus. Viral movement across the midgut is likely to be governed by a single major gene and modifying minor genes or a group of closely linked genes. These inbred mosquito lines will be useful in discovering the molecular basis for flavivirus resistance in Ae. aegypti.