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In Hymenoptera parasitoids, the reproductive mode is arrhenotoky, while a few species reproduce by thelytoky. The thelytoky of Hymenoptera parasitoids is generally genetically determined by the parasitoids themselves or induced by bacteria, including <i>Wolbachia</i>, <i>Cardinium</i>, and <i>Rickettsia</i>. <i>Diglyphus</i> <i>wani</i> (Hymenoptera: Eulophidae), a recently reported thelytokous species is a main parasitoid attacking agromyzid leafminers. To assess whether endosymbionts induce thelytoky in <i>D.</i> <i>wani</i>, we performed universal PCR detection and sequenced the V3-V4 region of 16S ribosomal RNA gene. In addition, bacteria were removed through high-temperature and antibiotic treatments, and the localized bacteria were detected using FISH. Based on general PCR detection, <i>Wolbachia</i>, <i>Cardinium</i>, <i>Rickettsia</i>, <i>Arsenophonus</i>, <i>Spiroplasma</i>, and Microsporidia were absent in laboratory and field individuals of thelytokous <i>D.</i> <i>wani</i>. Furthermore, 16S rRNA gene sequencing revealed that the dominant endosymbionts in thelytokous <i>D.</i> <i>wani</i> were not reproductive manipulators. High-temperature and antibiotic treatment for five consecutive generations cannot reverse the thelytokous pattern of <i>D.</i> <i>wani</i>, and no male offspring were produced. Moreover, no bacterial spots were found in the ovaries of <i>D.</i> <i>wani</i>. Thus, it is considered that the thelytoky of <i>D</i>. <i>wani</i> does not result in the presence of endosymbionts. This species is thus the second reported eulophid parasitoid whose thelytoky appears not to be associated with endosymbionts.