During mating, some male North American fireflies produce spermatophores from prespermatophores in their paired reproductive accessory glands. Other species of fireflies have neither prespermatophores nor spermatophores. To establish a pattern of spermatophore occurrence across firefly species, we examined the male internal reproductive system in 20 Japanese species belonging to 10 genera for the presence or absence of prespermatophores. Twelve species from seven genera produced prespermatophores, while eight species from three genera did not. Superimposed on a molecular phylogeny of Japanese fireflies based on mitochondrial 16S ribosomal DNA sequences, the basal group was prespermatophore producers. Prespermatophores appear to have been lost in two different lineages. Species without prespermatophores are characterized by degeneration of both the forewings and hindwings, and by body gigantism in females.