Using an F1 screen, 352 feral individuals of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were examined for the presence of Bacillus thuringiensis (Bt)-resistance alleles. These insects represented four geographical populations collected in central and northeastern Louisiana, USA, and one field population from the Gulf Coast area of Texas, USA, during 2006. The F1 screen used various crosses between field-collected insects and a laboratory strain of Cry1Ab-resistant D. saccharalis, including both reciprocal crosses and group mating. F1 neonates of the crosses were screened for Bt resistance on Bt maize leaf tissue. One field-collected individual of D. saccharalis was shown to have a Bt-resistance allele. Based on Bayesian analysis procedures, the Bt-resistance allele frequency in the five populations of D. saccharalis was 0.0028 with a 95% confidence interval of 0.0003–0.0079. The successful identification of a resistance allele in a field collection of insects suggests that the F1 screening technique could be an effective tool for detecting and monitoring rare Bt-resistance alleles in field populations of D. saccharalis.