The efficacy of sterile insect technique (SIT) depends on successful mating of released males with wild females. If the time of mating in a day of mass-reared and released males differs from those of wild females, the efficiency of SIT decreases. Therefore, understanding the molecular mechanisms controlling mating time of the target pests is particularly important for SIT. The period (per) gene, which has been considered as a key clock gene controlling the mating time of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), was cloned from two strains having different times of mating during the day. DNA sites varied in the 5′ and 3′ untranslated regions and at synonymous sites, although protein sequences were identical. We also provide phylogenetic relationships among PER protein sequences of dipteran species including several tephritid pest species. The functional domains of PER in the melon fly are very similar to those in other tephritid species. A luciferase reporter assay showed that the melon fly PER can functionally complement that of Drosophila melanogaster (Meigen). The results implicate that the major genetic cause of the difference in circadian periods, and thus in reproductive isolation, is probably one or more other clock gene(s). Thus, the series of studies may provide a novel factor concerning genetic quality control of mass-reared insect pests for SIT, which depends on successful mating of released males and wild females.