Three species of iso-female lines of Korean anophelines (Anopheles sinensis, An. lesteri, and An. sineroides) were identified based on combinative diagnostic characteristics of morphology, metaphase karyotypes, crossing experiments, and comparison of DNA sequences of ribosomal DNA Internal Transcribed Spacer 2 (ITS2) and further analyzed to determine their genetic relationships. Upon comparison of the F1-hybrids with those of their parent species as the control, differences in viabilities were observed. The viability of F1-hybrids with high larval mortalities, the failure of all pupae to emerge as adults, and the F1-hybrid's salivary gland polytene chromosomes showing complete asynapsis of all arms from the crosses between An. sinensis and An. lesteri, as well as An. sinensis and An. sineroides, suggested that these three cryptic species exhibited signs of an entirely reproductive isolation.