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 × 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 × C male crosses, the hybrid males showed partially sterile testes in which most spermatozoa were abnormal (enlarged head) and inactive, and they had very little success in inseminating females. The salivary gland polytene chromosomes of F₁ hybrid larvae from species C female × species E male showed a fixed heterozygous inversion on the 3L arm. Those hybrids F₁ from species E female × species C male showed partial asynapsis on identified arms (2R and 3L) and a fixed heterozygous inversion on the 3R arm. When the hybrid females from both directions of crosses were F₁ backcrossed with either C or E males, they produced male progeny with abnormal spermatozoa. Study of mating behavior in a 30 × 30 × 30-cm cage showed that the C males failed to mate with either C or E females, indicating that species C males cannot breed in confined spaces (lack stenogamy). Putative species E males had little success in inseminating species C females. This study provides strong evidence of genetic incompatibility between An. minimus species C and putative species E, supporting previous data that species E is a distinct species in the An. minimus complex.