Simple SummaryThe sterile insect technique is an environmentally-friendly method that can be used to manage populations of the South American fruit fly Anastrepha fraterculus, but its sterilizing doses have been determined only under normoxia. To maintain sterile male fly quality without jeopardizing sterility, a strategy of applying the radiation dose under an oxygen-reduced atmosphere is commonly used. Therefore, this study tested a range of gamma radiation doses under hypoxia on pupae from a bisexual strain and a novel genetic sexing strain (GSS) of A. fraterculus. Four types of crosses between irradiated flies under hypoxia and nonirradiated flies of the bisexual strain were set to assess sterility for each strain and radiation dose. For males from both strains, the effect of radiation dose on the percentage of egg hatch, egg-to-pupa recovery, and recovery of adults were determined. A dose of 74 Gy reduced egg hatch by 99% regardless of the male strain and was considered the sterilizing dose. The fertility of irradiated females and development of their offspring were severely affected, even at low doses under hypoxia, and no eggs were laid with doses above 50 Gy.A common strategy used to maintain sterile fly quality without sacrificing sterility is to irradiate the insects under an oxygen-reduced atmosphere. So far, sterilizing doses for the South American fruit fly Anastrepha fraterculus have only been determined under normoxia. Our study reports for the first time the dose-sterility response under hypoxia for two different A. fraterculus strains. The pupae were derived from a bisexual strain (a Brazilian-1 population) and a recently developed genetic sexing strain (GSS-89). Two hours prior to irradiation, pupae were transferred to sealed glass bottles and irradiated when oxygen concentration was below 3%. Four types of crosses with nonirradiated flies of the bisexual strain were set to assess sterility for each radiation dose. For males from both strains, Weibull dose–response curves between radiation doses and the proportion of egg hatch, egg-to-pupa recovery, and recovery of adults were determined. The GSS males revealed high sterility/mortality levels compared to males from the bisexual strain at doses < 40 Gy, but a dose of 74 Gy reduced egg hatch by 99% regardless of the male strain and was considered the sterilizing dose. The fertility of irradiated females was severely affected even at low doses under hypoxia.