Haplodiploidy is the sex determination mode of all Hymenoptera. Whereas most species reproduce by arrhenotokous parthenogenesis (males develop from unfertilized eggs and females from fertilized eggs), many reproduce by thelytokous parthenogenesis (unfertilized eggs develop into diploid females). Thelytoky can be determined genetically or by infection with maternally inherited endosymbiotic bacteria. To date, in the large Chalcidoidea superfamily, thelytokous parthenogenesis has almost always been associated to infection by microorganisms belonging to the genera Wolbachia, Cardinium and Rickettsia. A genetic mechanism for thelytokous chalcidoids has been suggested only for few species, as for Trichogramma cacoeciae. The greenhouse thrips Heliothrips haemorrhoidalis is an important worldwide pest, uniparental and thelytokous. Its endoparasitoid Thripoctenus javae is uniparental and thelitokous too. Main aim of this study is to test with an integrative approach if endosymbiotic bacteria are involved in T. javae thelytoky. Antibiotic and high temperature treatments were performed to investigate whether reproduction of T. javae could be reverted to arrhenotoky. FISH analyses with bacterial probes were performed on ovaries, and PCR screenings with universal and specific primers were carried out on whole insects to detect not only symbionts known to cause thelytoky in Chalcidoidea, but also additional sex manipulators such as Spiroplasma, Arsenophonus, and Microsporidia reported for other insects. Furthermore T. javae ITS2, 28S-D2 and mtDNA-COI genes were sequenced and used in phylogenetic inferences of Entedoninae subfamily and to test the homogeneity of the studied populations. No evidence of infection was found in T. javae parthenogenetic females and it was impossible to revert them to sexual reproduction by heat or antibiotic treatments. This makes T. javae one of the very few chalcidoids whose thelytokous reproduction is neither associated to presence of bacterial endosymbionts nor attributable to polyploidy as for its host; indeed, T. javae is known to have a normal diploid number of chromosomes (2n=12).