Global wheat production is seriously threatened by salinity stress, especially in irrigated and desert areas where crop yield is hampered by salt buildup in the soil. Utilising comparative transcriptome analysis of two genetically diverse wheat genotypes—a salt-tolerant and a salt-sensitive typetreated with glycine betaine (GB), the goal of this work is to discover important genes and pathways involved in the salt stress tolerance of wheat (Triticum aestivum L.). Due to its osmoprotective qualities, glycine betaine is known to increase plants' resistance to abiotic stressors like salt. Gene expression in wheat genotypes exposed to four treatment combinations—control, salt stress, glycine betaine therapy, and a combination of both stressors—was analysed using RNA sequencing. In our research, we found thousands of differentially expressed genes (DEGs) between the salt-tolerant and sensitive genotypes, underlining important pathways associated with oxidative stress, ion transport, and osmotic adjustment. Treatment with glycine betaine changed the expression of genes that respond to stress, especially when exposed to salt stress, which enhanced the mechanisms used for stress adaption. Important biological processes that are essential for stress tolerance were found using GO (Gene Ontology) enrichment analysis. These processes include cell wall metabolism, fatty acid production, and cytoskeletal dynamics. These results give molecular insights into the mechanisms by which glycine betaine increases wheat resilience to salinity, potentially serving as targets for agronomic and genetic approaches aimed at enhancing wheat performance in areas impacted by salt.