Viral determinants involved in systemic infection of hosts by monocot-infecting plant viruses are poorly understood. Wheat streak mosaic virus (WSMV, genus Tritimovirus, family Potyviridae) exclusively infects monocotyledonous crops such as wheat, oat, barley, maize, triticale, and rye. Previously, we reported that WSMV CP amino acids 36–84 are expendable for systemic infection of wheat, maize, barley and rye. In this study, the role of coat protein (CP) in systemic infection of oat by WSMV was examined by using a series of viable deletion mutants. WSMV bearing deletions within or encompassing all of amino acids 36–57 efficiently infected oat, indicating that these amino acids are dispensable for systemic infection of oat. However, WSMV mutants lacking CP amino acids 58–84 or 85–100 failed to systemically infect oat. Furthermore, green fluorescent protein-tagged WSMV mutants lacking CP amino acids 58–100 elicited local foci in oat but failed to enter the vasculature. These data suggest that CP amino acids 58–100 are required for systemic infection of oat by WSMV by specifically facilitating virus long-distance transport in oat.