Eldana saccharina is a major stalk boring pest of sugarcane in South Africa, causing substantial yield and revenue losses. Developing sugarcane cultivars with durable resistance to E. saccharina is therefore a priority for breeding programmes. In this study, we employed RNA sequencing (RNA-seq) to characterise the transcriptional responses to E. saccharina by comparing infested plants with uninfested controls in sugarcane genotypes displaying contrasting resistance phenotypes. Differential expression analysis, combined with functional annotation, revealed clear and contrasting defence responses between resistant and susceptible genotypes. Distinct sets of co-regulated genes were identified, reflecting genotype-specific immune activation patterns. The detection of both early and late transcriptional responses highlights how the timing and coordination of gene expression contribute to resistance or susceptibility outcomes. Integrating these expression signatures identified a candidate region harbouring a gene that was strongly induced in resistant genotypes following infestation. This genomic region exhibited presence/absence variation (PAV) among the genotypes analysed, enabling the development of a robust PCR-based marker for reliable tracking of the locus across diverse germplasm. The marker was subsequently assessed by screening genotypes from a late-stage breeding trial. The analysis revealed an association between the locus presence and enhanced resistance to E. saccharina. Collectively, these findings provide novel molecular insight into the sugarcane-E. saccharina interaction and identify a resistance-associated PAV locus that can be deployed as a practical PCR-based defence-response marker. This marker offers a useful tool to accelerate selection of resistant genotypes through marker-assisted breeding.