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Abstract The fall armyworm ( Spodoptera frugiperda ) is a highly polyphagous lepidopteran pest of relevant food and fiber staple crops. In the Americas, transgenic corn and cotton producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have controlled and reduced the damage caused by S. frugiperda . However, cases of field-evolved S. frugiperda resistance to Bt corn producing the Cry1F insecticidal protein have been documented in North and South America. When characterized, field resistance to Cry1F is linked to insertions and mutations resulting in a modified or truncated ABC transporter subfamily C2 ( SfABCC2 ) protein that serves as Cry1F receptor in susceptible S. frugiperda . In this work, we present detection of a large genomic deletion (~ 8 kb) affecting the SfABCC2 and an ABC transporter gene subfamily 3 –like gene ( SfABCC3 ) as linked to resistance to Cry1F corn in a S . frugiperda strain from Florida (FL39). Monitoring for this genomic deletion using a discriminatory PCR reaction in field-collected S. frugiperda moths detected individuals carrying this allele in Florida, but not in surrounding states. This is the first report of a large genomic deletion being involved in resistance to a Bt insecticidal protein.