Sawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria (Wolbachia and Cardinium) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the presence of Cardinium, 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini, both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults.