Cultivar resistance is a key management strategy for the sugarcane borer, Diatraea saccharalis (F.), the primary pest in Louisiana sugarcane, but mechanisms of resistance are not well understood. This research evaluated the potential mechanisms of cultivar resistance to D. saccharalis among commercially produced sugarcane cultivars and experimental lines through three field screenings, two greenhouse experiments, and one diet incorporation assay. The resistant standards HoCP 85-845, HoCP 04-838, and L 01-299 were among the cultivars with the lowest D. saccharalis injury levels in both field and greenhouse trials. Cultivars HoCP 00-950 and L 12-201 were among the most heavily injured in both trials. Differences in oviposition among cultivars in the greenhouse choice study were not detected, suggesting adult preference is not a key factor in resistance. This was also supported by the no-choice greenhouse experiment in which up to 9-fold differences in neonate establishment among cultivars were detected. Larval injury among cultivars in greenhouse experiments was consistent with field studies suggesting traits that affect neonate establishment (e.g., rind hardness) help to confer resistance in the field. In the diet incorporation assay, lower larval weights and longer time to pupation were observed on resistant cultivar Ho 08-9003, but no differences were found among current commercial cultivars. Continuous evaluation of cultivar resistance to D. saccharalis is important in developing effective integrated pest management strategies for this pest. More research into plant characteristics (e.g., leaf sheath tightness and pubescence) associated with resistance is needed.