The grain aphid Sitobion avenae (Fabricius) is one of the most destructive pest of wheat. The use of resistant wheat germplasm appears to be an excellent solution to this problem. Elite bread wheat cultivars in China have only limited resistance to this pest. The present study was carried out to investigate the potential of a tetraploid wheat variety Lanmai, showed high resistance to S. avenae at both seedling and adult plant stages, to serve as a source of resistance genes. Lanmai has been shown to display antixenosis and antibiosis based on adult fecundity tests and choice bioassays. Suppression subtractive hybridization (SSH) was employed to identify and isolate the putative candidate defense genes in Lanmai against S. avenae feeding. A total of 134 ESTs were identified and categorized based on their putative functions. BLAST results showed that 112 ESTs (83.58%) were homologous to previously characterized proteins of Triticum crops. They were identified to have functions related to various biological processes, including defense and defense signaling (14.9%), photosynthesis and energy (14.9%), protein synthesis (14.2%), protein protein degradation (6.7%), metabolism (11.9%), signal transduction (9.7%), cell organisation and division (3.0%), transcription (6.0%), cell wall (2.2%) and transport (9.0%). Quantitative RT-PCR analysis of 30 selected genes confirmed their differential expression over time between the resistant wheat cultivar Lanmai and susceptible wheat cultivar Polan305 during S. avenae infection. There were eleven genes related to the photosynthesis process, and only three genes were observed to have higher expression in Lanmai than in Polan305 after S. avenae feeding. Gene expression analysis also revealed that Lanmai possessed distinct regulation of salicylic acid and jasmonic acid pathways after S. avenae infestation. This study provided further insights to understand the role of defense signaling networks in wheat’s resistance to S. avenae and indicates that the resistant tetraploid wheat variety Lanmai may provide a valuable resource in breeding wheat for resistance to aphids.