Some viral proteins were translated in cap-independent manner via internal ribosome entry site (IRES), which ever maintained conservative characteristic among different isolates of same species of virus. However, IRES activity presented 7-fold of variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and hepta-nucleotide top loop of H2 in LYJN isolate synergistically ensured the higher IRES activity than that in HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. RNA2 5′-UTR specifically interacted with the wheat eIF4E, which was accomplished by the top loop of H1 in HC isolate or the top loop of H1 and H2 in LYJN isolate. Different IRES activity of WYMV RNA2 was regulated by different numbers of eIF4E-binding site and their synergistic effect, which was accomplished by the proximity of H1 and H2 due to the flexibility of middle stem in H1. It is represented a novel evolution pattern of IRES.