Quantitative trait loci (QTL) affecting the ability of the Aedes aegypti midgut to become infected with Dengue 2 virus (DEN2) were mapped in the F5 generation of an advanced intercross line (AIL). A strain of Ae. aegypti previously selected for DEN2 susceptibility was crossed to a new strain selected for refractoriness to midgut infection. In P1 and F1 parents and 147 F5 offspring, genotypes at forty-four cDNA loci were analysed. A new sex linked QTL and a second QTL on chromosome II with genotypes subject to balancing selection were detected that condition midgut susceptibility. Alleles at these QTL contributed additively in determining susceptibility and accounted for approximately 24% of the phenotypic variance. Markers associated with a midgut escape barrier were inconsistently supported.