Transgenic corn,Zea mays(L.), expressing insecticidal toxins such as Cry1Fa, fromBacillus thuringiensis(Bt corn) targetingOstrinia nubilalis(Hübner) (Lepidoptera: Crambidae) resulted in over 20 years of management success. The first case of practical field-evolved resistance byO. nubilalisto a Bt corn toxin, Cry1Fa, was discovered in Nova Scotia, Canada, in 2018. Laboratory-derived Cry1Fa-resistance byO. nubilaliswas linked to a genome region encoding the ATP Binding Cassette subfamily C2 (ABCC2) gene; however, the involvement ofABCC2and specific mutations in the gene leading to resistance remain unknown. Using a classical candidate gene approach, we report onO. nubilalis ABCC2gene mutations linked to laboratory-derived and field-evolved Cry1Fa-resistance. Using these mutations, a DNA-based genotyping assay was developed to test for the presence of the Cry1Fa-resistance alleles inO. nubilalisstrains collected in Canada. Screening data provide strong evidence that field-evolved Cry1Fa-resistance inO. nubilalismaps to theABCC2gene and demonstrates the utility of this assay for detecting the Cry1Fa resistance allele inO. nubilalis. This study is the first to describe mutations linked to Bt resistance inO. nubilalisand provides a DNA-based detection method that can be used for monitoring.