The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F₂ screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10⁻³) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F₂ screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.