Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) or “Qfly,” is the most serious horticultural pest in Australia, with a bioclimatic range that extends from the tropical north to the temperate south. Various Australian horticultural exports depend on certification that they originated from B. tryoni-free areas. To eliminate, rather than suppress, B. tryoni in production areas, a sterile insect technique (SIT) campaign directed at B. tryoni has been in operation in southeastern Australia since 1997. Like many other SIT programs around the world, the B. tryoni SIT program relies on fluorescent dust to mark the sterile insects. However, fluorescent dust marking does not provide 100% accuracy in the identification of sterile insects, as required where the aim is to declare regions completely free of fruit fly. Here, we show that novel mitochondrial markers can be introduced into a strain of B. tryoni by interspecies hybridization between B. tryoni and a related but well-differentiated species, Bactrocera jarvisi (Tryon), followed by backcrossing of the hybrid strain with the parental B. tryoni strain. These novel markers do not affect the viability of the strain as measured by pupation and eclosion rates. A simple polymerase chain reaction-based test is described that distinguishes the marked B. tryoni from wild B. tryoni. As required in practice, the test was shown to work reliably on DNA extracted from dead flies that had remained in field traps for up to two weeks.