Evaluation of host-plant resistance on sugarcane to the sugarcane stem borers of Diatraea spp. is normally conducted in Colombia under field conditions, where environmental variations make the study of the insect–plant relationships difficult. Additionally, several species (i.e., D. saccharalis, D. indigenella, D. tabernella, and D. busckella), which are predominant in Colombia, can overlap in their distribution, raising the question of whether different varieties have the same responses to different pest species. The present study conducted evaluations of host-plant resistance under screen house conditions using two contrasting varieties (CC 93-3895, resistant, and CC 93-3826, susceptible) that were infested with the above-mentioned borer species. Observations of pest injury were conducted on internodes, leaves, and spindles. Survival and size (body mass) of the individuals recovered were analyzed and a Damage Survival Ratio (DSR) was proposed. The resistant CC 93-3895 exhibited less stalk injury, less emergence holes on internodes, and lower DSR; additionally, recovery of pest individuals was lower in comparison with CC 93-3826, independent of the borer species. Insect–plant interactions are discussed, as no previous information was available for three of the species tested (i.e., D. tabernella, D. indigenella, and D. busckella). This screen house protocol is proposed to characterize host-plant resistance among several cultivars from the Colombian sugarcane germplasm bank, using CC 93-3826 and CC 93-3895 as contrasting controls and D. saccharalis as the species model.