The sugarcane borer, Diatraea saccharalis (F.), is a dominant corn stack boring pest and a major target of Bacillus thuringiensis (Bt)-corn in many areas of the mid-southern region of the United States. A Cry1Ab-resistant strain of D. saccharalis, capable of survival on commercial Bt corn plants, was established using an F2 screening procedure. Larval survival of Cry1Ab-resistant, -susceptible, and -heterozygous genotypes was evaluated on five non-Bt and seven Bt field corn hybrids at two plant stages. During the vegetative stages, all seven Bt corn hybrids were highly efficacious against the three genotypes, while 8-18% of the heterozygous genotype survived on reproductive stage plants for four Bt corn hybrids. Susceptibilities of Cry1Ab-susceptible and -resistant strains were evaluated for four Bt proteins: Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. LC50 values of the Cry1Ab-resistant strain were >80-, 45-, 4.1-, and -0.5-fold greater than that of the susceptible strain to the four proteins, respectively. Relative fitness on non-toxic diet, diet treated with low concentrations of Cry1Ab toxin, and on conventional corn plants was compared for five genotypes of D. saccharalis. Larvae of Cry1Ab-susceptible and Cry1Ab-resistant strains on both non-toxic diet and non-Bt corn plants developed normally. There were no significant differences between the two strains in all measured biological parameters, suggesting a lack-of-fitness cost of the Cry1Ab resistance in D. saccharalis. Larval development, growth, and survival of the Cry1Ab-susceptible strain were significantly affected on diet treated with low concentrations of Cry1Ab toxin, while the effect to the resistant strains was not/or less significant. Using various genetic crosses, inheritance of Cry1Ab resistance in D. saccharalis was assessed on Bt corn leaf tissue, intact Bt corn plants, and diet containing Cry1Ab toxin. Cry1Ab resistance in D. saccharalis was inherited as a single autosomal gene. The resistance was incompletely or nearly completely recessive on Bt corn leaf tissue and intact Bt corn plants, while the dominance increased as Cry1Ab concentrations decreased when it was tested on Cry1Ab-treated diet. Results generated from this study will provide valuable information in understanding Bt resistance mechanisms and developing effective strategies for managing resistance of D. saccharalis to Bt corn.