Inter- and intraspecific differences between Asteralobia gall midges (Diptera: Cecidomyiidae) that induce axillary bud galls on several Ilex species were studied from morphological, molecular, and ecological points of view. Asteralobia sasakii (Monzen) and Asteralobia soyogo (Kikuti) were clearly distinguished from one another by thoracic setal and scale counts and by sequences of partial mitochondrial DNA cytochrome oxidase subunit I region. Because adults of A. sasakii emerged within a shorter period of time and later in the season than those of A. soyogo, they were considered to be temporally isolated. The host ranges of A. sasakii and A. soyogo did not overlap. A. sasakii was confirmed to use Ilex crenata Thunberg, Ilex crenata var. paludosa (Nakai) Hara, and Ilex maximowicziana Loesener for galling, and A. soyogo included galls on Ilex chinensis Sims, Ilex integra Thunberg, Ilex leucoclada (Maximowicz) Makino, and Ilex pedunculosa Miquel. Molecular phylogenetic analysis revealed that host races were not formed either in A. sasakii or in A. soyogo populations, whereas geographical variations were detected for both species at the DNA level. The haplotype diversity was significantly higher in A. soyogo than in A. sasakii. The DNA analysis suggested that A. sasakii and A. soyogo had speciated allopatrically. On the basis of the current study, we concluded that A. soyogo had been already established in Honshu, Shikoku, and Kyushu, Japan, when A. sasakii arrived in these areas from the southwest islands more recently.