DNA double-strand breaks (DSBs) are one of the most biologically significant DNA damage lesions. Exposure to ionizing radiation (IR) causes DSBs in living organisms, which trigger intrinsic DNA repair mechanisms. Phosphorylation of the C-terminal of the core histone protein H2AX (termed γH2AX when phosphorylated) is an early known response to DNA DSBs. Quantification of the γH2AX response offers a highly sensitive and specific assay for detecting DSB formation and repair. Postharvest exposure to IR of 150–400 Gy is an increasingly prominent phytosanitary measure in a variety of Australian (and imported) fruit. The radiation-induced γH2AX response has been shown to be highly persistent in the Queensland fruit fly (“Q-fly”; Bactrocera tryoni), Australia’s most economically damaging insect pest of horticultural crops, lasting at least 17 days after exposure to IR. The presence of persistent γH2AX, indicating ongoing repair of impaired DNA, can be used to assess irradiation exposure in fruit flies. A direct and reliable assay using γH2AX as a marker of prior IR exposure in fruit flies has the potential to facilitate domestic and international trade in commodities that have been irradiated for disinfestation.