Sugarcane smut, caused by the fungus Sporisorium scitamineum, is the most challenging disease of sugarcane, causing significant losses in cane yield. There is a dearth of information on smut resistance mechanism in elite parents for the development of smut-resistant varieties. In the present study, we adopted joint Illumina- and Single Molecule Real-Time (SMRT)-based RNA-seq analysis to identify transcript expression in an smut-resistant and -susceptible parents (ROC25 and ROC22) infected with S. scitamineum. A total of 79,885 high-quality transcripts was obtained, including 60,115 open reading frames, 3692 alternate splicing isoforms, 1799 long non-coding RNAs, 29,139 simple sequence repeats, and 7794 transcription factors. About 92.72% of the total transcripts were annotated, which should have increased the available data amount for transcriptome profile analysis. There were 2033 and 9716 differentially expressed transcripts (DETs) in ROC22 and ROC25, respectively. The analyses of GO and KEGG enrichment showed that more GO terms and KEGG pathways were observed in ROC25 than ROC22. It was found that MAPK signalling pathway-plant, phenylpropanoid biosynthesis, plant-pathogen interaction, linoleic acid metabolism, and starch and sucrose metabolism were enriched both in resistant and susceptible parents. In addition, MAPK superfamily genes were differentially regulated in different parents, more DETs of MEKK1 and MKK4 were detected in resistant parent, and the relative expression levels of MKK5, MPK10, and MPK12 genes were specifically altered in resistant parent. It suggested that MAPK superfamily genes might play the important roles in the regulation of sugarcane response to S. scitamineum infection. Moreover, lots of transcription factors (TFs) associated with plant disease resistance were found to respond to S. scitamineum infection in both ROC22 and ROC25 parents, including WRKY, MYB, NAC, and AP2/ERF-ERF. Majority of the TFs were up-regulated. Compared to the susceptible ROC22 parent, the number of activated transcription factors in the resistant ROC25 parent was higher, indicating that these extra TFs might have positive effects in the defense against S. scitamineum. This study provides a comprehensive set of reference transcripts for sugarcane and thus increases our understanding on the interactions between sugarcane and S. scitamineum, which should be helpful in guiding on exploitation and utilization of smut-resistance gene resources.