Insects exposed to genetically modified crops expressing Bacillus thuringiensis (Bt) toxins are under intense selection pressure that could result on widespread Bt resistance. Screening for early indications of Bt resistance developing in targeted Lepidoptera is conducted in many of the regions where genetically modified cotton and corn have been commercialized. Heliothis virescens (F.) (Lepidoptera: Noctuidae) has been selected in the laboratory to have a gene for resistance to Cry1Ac. We used this laboratory line to test the assumptions and theoretical predictions related to detection of recessive Bt-resistant alleles in field populations based on a second generation (F₂) screen. By creating single-pair families from mating a heterozygous Cry1Ac-resistant moth with a Cry1Ac-susceptible moth, we simulated the most common genotype when Bt-resistance alleles are at low frequency in the field. The second generation (F₂) neonates of single-pair families were screened daily with diagnostic concentration bioassays. Cry1Ac-resistant homozygous larvae were detected, but the proportion of resistant larvae was generally below the theoretical expectation of 6.25% and was influenced by the moth F₁ sib-mating density and by the day of oviposition of F₂ eggs. Logistical considerations such as F₁ sib-mating density and F₂ neonate screening are important for the successful implementation of a reliable method.