Grapevine downy mildew, caused by Plasmopara viticola, significantly damages vineyards and is one of the most devastating diseases affecting cultivated grapes worldwide. In this study, we characterized the phenotypic and molecular traits of 11 P. viticola isolates from four grape-growing regions in South Korea. Additionally, we investigated the diversity of pathogenicity among these isolates and conducted an assay to evaluate the response of grape cultivars to P. viticola infection. Lemon-shaped sporangia were identified in the collected isolates, which released zoospores into the suspension at room temperature. Within a few hours of inoculation, the zoospores developed germ tubes. We tested 11 P. viticola isolates for pathogenicity in 845 grape cultivars to screen for grape host resistance to downy mildew infection. Among the tested isolates, JN-9 showed the highest virulence. Grape cultivars displayed varying phenotypic reactions to P. viticola infection: approximately 7% were highly susceptible, 41% were susceptible, 20% were moderately susceptible, 8% were resistant, and 24% exhibited extreme resistance. Phylogenetic analysis based on four genomic regions (internal transcribed spacer 1 [ITS1], actin, beta-tubulin, and cytochrome c oxidase II) revealed a close evolutionary relationship among all the Korean isolates, forming a single monophyletic lineage. Notably, these isolates showed greater similarity to European isolates than to American isolates. This comprehensive study contributes to a deeper understanding of the identity and behavior of P. viticola, which is crucial for developing effective resistance strategies against this pathogen in grape cultivars cultivated in South Korea.