Phomopsis viticola is the causal agent of Phomopsis cane and leaf spot on Vitis spp., a persistent and economically important disease in temperate regions. Here we describe the transformation of this fungus with two different constructs (pBHt2_sGFP and pIGPAPA) containing the green fluorescent protein (GFP) and the hygromycin B resistance gene (hph). Protoplast-mediated transformation yielded mitotically stable transformants with no change in virulence on grape internodes and leaves in comparison to the wild type. These transformants will be critical tools for elucidating fungal penetration of host plants, invasive growth and the nature of its host association.