Differentially expressed genes help in exploring plant defense mechanism under variable stress conditions. In current investigation, RNA sequencing was executed to explore the differential gene expression in resistant and susceptible varieties of Cotton (Gossypium hirsutum), upon infection with Aspergillus tubingensis. Comparative RNA-Seq of control and infected plants was performed using Illumina HiSeq 2,500. Overall 79.84 G clean data was generated and 6,558 DEGs were identified in both varieties, in response to pathogen inoculation. Differentially expressed genes were found to be involved in defense, antifungal response, signaling pathways, oxidative burst and transcription. Genes involved in defense responses, MAPK signaling, cell wall fortification and signal transduction were highly induced in resistant variety. Real time PCR also revealed the up regulation of MAPKKK YODA like, L-ascorbate oxidase, late embryogenesis abundant protein (At1g64065) and flavonoid 3',5'-hydroxylase-like, in resistant variety. Elevated accumulation of such DEGs in resistant variety could function as the source for identifying biomarkers for breeding and these can be used as potential candidate genes for transgenic manipulation. Their study also helped in understanding complex plant-fungal interaction and advanced the understanding of plant-microbe interaction. Inclusively, our findings provide an indispensable foundation for advanced understanding of the plant resistance mechanisms of cotton.