Anopheles sinensis and An. pullus, the 2 predominant anopheline mosquito species in Korea, are major malaria vectors. Their morphological similarity makes them difficult to distinguish, and their taxonomic relationship remains unclear. Kanda and Oguma produced fertile F₁ An. sinensis × An. pullus hybrids, suggesting a conspecific relationship. However, our recent internal transcribed spacer 2 DNA sequence comparison of these taxa revealed 32.9% divergence, suggesting that An. sinensis and An. pullus may be distinct species. In the present study, we reassessed the relationship of An. sinensis and An. pullus in hybridization experiments. Laboratory-raised isofemale lines of An. sinensis form B and An. pullus form A and B were reciprocally crossed. The 3 isofemale lines were established from fully engorged females caught from the wild and morphologically identified as An. sinensis or An. pullus. The isofemale lines represented 3 distinct metaphase karyotypes: An. sinensis form B (X, Y₂) and An. pullus form A (X₁, Y₁) and form B (X₂, Y₂). The F₁ hybrid larvae showed low viability and completely asynaptic salivary gland polytene chromosomes. The subset of F₁ hybrids that developed to adults showed sex-ratio distortion; ovarian follicles developed abnormally in females, and males suffered atrophy of accessory glands and testes. These results strongly suggest that the Korean morphological species An. sinensis and An. pullus are biological species, separated by complete postzygotic reproductive isolation.