F2 neonates of 213 iso-line families of the sugarcane borer, Diatraea saccharalis (F.), were screened for Bt resistance on Bt corn leaf tissues dissected from fully-expanded leaves of V6-V8 stage DK69-70 Bt corn plants containing the Cry1Ab gene. Four families survived the Bt corn leaf tissues after 10 d and three families survived after 15 d. One family was confirmed to carry a major Bt resistance allele. The resistant D. saccharalis survived and completed larval development on DK69-70 Bt plants in the greenhouse. The resistance allele detected in this D. saccharalis population is the first major Bt resistance allele found in corn stalk borers. It is also the first major allele detected with F2 screen that resist to a commercial Bt plants. The unexpected major resistance allele observed in this Louisiana D. saccharalis population suggests that management of D. saccharalis resistance to Bt corn is important to ensure the long-term success of Bt corn for management of this corn stalk borer pest.