Hartigiola faggalli (Monzen), a cecidomyiid species that induces leaf galls on Fagus crenata Blume (Fagales: Fagaceae), was studied to assess the degree of sexual isolation between known intraspecific populations derived from two different gall types. “Upper-type galls” form on the lateral veins of upper leaf surfaces, whereas “lower-type galls” develop between the lateral veins of lower leaf surfaces. The two populations were distinguished based on slight differences in theirDNAsequences. They coexisted in F. crenata forests. Emergence, swarming, mating, and oviposition occurred sequentially each day and almost simultaneously in both populations. Thus, they were not isolated from each other in time or space. However, 85% of 134 swarming males flew to females of the same population when responding to female sex pheromone. About 92% of 251 mating pairs were homogenic, and IPSI indicated a significantly homogenic mating. The female sex pheromone and male sensitivity to the pheromone seemed to differ between the two populations. After mating, females of each population oviposited their eggs only on either the upper or lower surfaces of fresh leaves. The strongly assortative mating combined with differences in pheromones and gall morphology indicates that the two populations are almost completely reproductively isolated and that they have diversified into the stage of sibling species.