Simple SummaryTephritid flies, including the Mexican fruit fly Anastrepha ludens, are key agricultural pests responsible for billions of dollars of damage each year due to the female flies which lay eggs and develop maggots in the fruits of hundreds of species of economically-important plants. Integrated pest management practices such as the sterile insect technique were developed which rely on the mass rearing and release of millions of sterile males of the same pest species in order to suppress the pest reproductive capacity. The presence of females in early bisexual strains often caused high rearing and release costs plus damage to crops by oviposition. Therefore, using classical genetic tools, genetic sexing strains were developed for some fruit flies in order to produce male-only colonies which enhanced the technique efficacy, cost-efficiency and reduced undesirable side-effects such as fruit damage by the mass reared females. In this paper the process for the development and characterization of a new genetic sexing strain (GSS) for A. ludens is documented, using the low-dose irradiation and line selection techniques. The new GUA10 GSS, when compared with its predecessor Tapachula-7, promises to further increase the sterile insect technique performance given its higher quality, yield and genetic stability.Anastrepha ludens (Loew) is one of the most destructive insect pests damaging several fruits of economic importance. The sterile insect technique (SIT) is used under an area-wide integrated pest management approach, to suppress these pest populations. Mass rearing facilities were initially established to produce sterile males of bi-sexual strains in support of SIT. The first genetic sexing strain (GSS) for A. ludens, Tapachula-7, based on pupal color dimorphism, was a key development since the release of males-only significantly increases the SIT efficiency. In this study, we document the development of a novel pupal color-based GSS. Twelve radiation-induced translocation lines were assessed as potential GSS in terms of recombination rates and rearing efficiency at a small scale. The best one, GUA10, was cytogenetically characterized: it was shown to carry a single translocation between the Y chromosome and chromosome 2, which is known to carry the black pupae marker. This GSS was further evaluated at medium and large scales regarding its genetic stability, productivity and quality versus Tapachula-7. GUA10 presented better genetic stability, fecundity, fertility, production efficiency, flying ability, and male mating, clear indicators that GUA10 GSS can significantly improve the efficacy and cost-effectiveness of SIT applications against this pest species.