AbstractTo establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant‐infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double‐fluorescent (green and mCherry) protein‐tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N‐terminus (amino acids 1–97), the core (amino acids 98–245), and the C‐terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N‐terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell‐to‐cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans‐expression of the wild‐type CP either by TuMV or through genetic transformation‐based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis‐expressed CP is functional.