Wolbachia are cytoplasmically inherited bacteria that are reported to infect at least 18–30% of all insect species. Our survey of Lepidoptera indicated that 44.9% of forty-nine species and 77.8% of nine families tested positive for Wolbachia using PCR with wsp primers. Nineteen species had not been described previously as infected. In particular, although Pieris rapae, which is a common species in Japan, is infected by Wolbachia, the prevalence was very low (3.4%) and there were some localities where Wolbachia could not be detected. The probability of detection of Wolbachia depends on the number of screened individuals of P. rapae. The results indicate that the actual number of species that are positive for Wolbachia may be higher than previously reported.