The major malaria vector Anopheles funestus belongs to a group of morphologically similar species that are commonly distinguished from one another through the use of chromosomal and molecular techniques. Indoor resting collections of mosquitoes from Malawi were initially identified as An. funestus by morphology, but failed to have this confirmed by the species-specific polymerase chain reaction assay. Sequence analysis of the internal transcribed spacer region 2 identified variations within the An. funestus-specific primer binding site and showed a sequence variation of 4.5% compared with An. funestus. Domain 3 analysis showed sequence variation of 1.5% from An. funestus. Cytogenetic analysis of the polytene chromosome banding arrangements showed that the specimens were homosequential with An. funestus, with fixed inverted arrangements of the 3a, 3b, and 5a inversions commonly polymorphic in An. funestus. The chromosomes of hybrid females showed levels of asynapsis typical of inter-species crosses. These molecular and cytogenetic observations support the conclusion that this Malawi population is a new species and it has provisionally been named An. funestus-like.