Fusarium head blight (FHB), mainly caused by Fusarium graminearum (Fg), is a devastating fungal disease in wheat that causes significant damage by compromising both the yield and the quality. In addition, mycotoxin contamination in the infected grains can cause severe health risks to both humans and animals. Although there are useful genetic sources of FHB resistance in common wheat, this is very limited for durum wheat. To explore and identify useful genetic variations that promote resistance, we analyzed disease responsive transcriptome data from durum wheat to determine the gene expression levels under fungal infection. A greenhouse experiment was carried out at Fargo, North Dakota over three years (2022, 2023 and 2024), with six durum lines with differences in resistance against FHB. Lines were subjected to transcriptomic study under the disease pressure to find the DEGs and explore the possible pathways involved in conferring resistance against this disease. The related durum germplasm, contain varying percentages of genetic sequence from resistant source Sumai3 background and correspondingly, display different levels of resistance against FHB. Spikes collected five days after inoculation with milli-Q water as mock and mixed race of Fusarium as treatment were used for RNA extraction and RNA-seq analyses. Sequencing information along with disease scores were used to perform differential expression study with the Hisat2-Stringtie-Ballgown (newer “tuxedo suite”) pipeline. In this study, we evaluated the pairwise comparisons between inoculated compared to mock, lines with or without Fhb1 gene and resistant reaction compared to susceptible reaction. A large number of DEGs were obtained for each condition (25,052 for inoculated vs mock, 12,605 for lines with vs without Fhb1 gene and 11,175 for resistant vs susceptible). Gene ontology evaluation showed that several defense responsive genes were being activated and highly enriched under disease pressure. In addition, several stress signaling pathways were being highly expressed along with cell wall reinforcement, different catalytic processes and pathways in response to biotic stimulus. KEGG pathway analysis revealed that many of the genes identified are part of several plant-pathogen interaction pathways, the MAPK signaling pathway and several metabolites biosynthesis pathways. STRING protein-protein interaction revealed the complex interconnectivity network across different proteins. Our current study revealed the important genes and their corresponding chromosomal coordinates. In the future functional analysis and further haploblock study will give an insight into fractions of DEGs that are associated with Sumai3 or P1277012 background and broaden our understanding on functioning of major FHB resistance genes.