Rhopalosiphum padi L. is one of the predominant aphids affecting wheat crops worldwide. Therefore, the identification of resistant genotypes and the understanding of molecular response mechanisms involved in wheat resistance to this aphid may contribute to the development of new breeding strategies. In this study, we evaluated the resistance of 15 wheat cultivars to R. padi and performed morpho-histological and gene expression analyses of two wheat cultivars (BRS Timbaúva, resistant and Embrapa 16, susceptible) challenged and unchallenged by R. padi. The main findings of our work are as follows: 1) most Brazilian wheat cultivars recently released are resistant to R. padi; 2) Green leaf volatiles are probably involved in the resistance of the BRS Timbaúva cultivar to the aphid; 3) trichomes were more abundant and larger in the resistant cultivar; 4) the internal morphology did not show differences between cultivars; 5) the lipoxygenase-encoding gene was downregulated in the susceptible cultivar and basal expression remained level in the resistant cultivar; and 6) the expression of resistance-related proteins was induced in the resistant but not in the susceptible cultivar. Lipoxygenase is the first enzyme in the octadecanoic pathway, a well-known route for the synthesis of signaling molecules involved in the activation of plant defense. The overall analyses suggest that the key steps in BRS Timbaúva resistance to R. padi may be presence or absence of green leaf volatiles decreasing the aphid preference and the action of nonglandular trichome as a physical barrier, which allows continuous lipoxygenase-encoding gene expression.