In the Anopheles minimus complex, 2 sibling species (A and C) are generally accepted. Recently, a 3rd species, provisionally designated An. minimus species E, has been described from the Ryukyu Archipelago, Japan, based on crossing experiments (A and E), DNA analysis, mitotic karyotypes, and some morphological characteristics. The present study reports the results of crossing experiments between species C and putative species E. Hybridization between the progeny of An. minimus species C from Thailand and putative species E from Japan revealed postzygotic genetic incompatibility. Although F1 hybrid progeny were obtained from both directions of crosses, the hybrid males from C female x E male crosses were completely sterile, with atrophied testes and accessory glands. In addition, the external terminalia of all of these males never completely rotated and the males failed to copulate by artificial mating. In E female x C male crosses, the hybrid males showed partially sterile testes in which most spermatozoa were abnormal (enlarged head) and inactive,