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Fusarium verticillioides is the pathogen associated with pokkah boeng disease (PBD), the most significant airborne disease of sugarcane. The molecular mechanisms that regulate the defense responses of sugarcane towards this fungus are not yet fully known. Samples of 'YT 94/128' (resistant, R) and 'GT 37' (susceptible, S) inoculated with F. verticillioides on the 14 days post-inoculation were used to analyze the transcriptome to screen R genes. In total, 80.93 Gb of data and 76,175 Unigenes were obtained after assembling the sequencing data, and comparisons of Unigenes with NR, Swiss-prot, KOG, and KEGG databases confirmed 42,451 Unigenes. The analysis of differentially expression genes (DEGs) in each sample revealed 9092 DEGs in 'YT 94/128,' including 8131 up-regulated DEGs and 961 down-regulated DEGs; there were 9829 DEGs in 'GT 37,' including 7552 up-regulated DEGs and 2277 down-regulated DEGs. The identified DEGs were mainly involved in catalytic enzyme activity, cell protease, hydrolytic enzymes, peptide enzyme, protein metabolism process of negative regulation, phenylpropanoid metabolism, extracellular region, aldehyde dehydrogenase, endopeptidase, REDOX enzyme, protein kinases, and phosphoric acid transferase categories. KEGG pathway clustering analysis showed that the DEGs involved in resistance were significantly related to metabolic pathways of phenylpropanoid biosynthesis, cutin, suberine and wax biosynthesis, nitrogenous metabolism, biosynthesis of secondary metabolites, and plant-pathogen interactions. This application of transcriptomic data clarifies the mechanism of interactions between sugarcane and F. verticillioides, which can help to reveal disease-related metabolic pathways, molecular regulatory networks, and key genes involved in sugarcane responses to F. verticillioides.