Grapevine leaf rust (GLR) caused by Phakopsora euvitis is a serious disease that results in severe leaf necrosis and early plant defoliation. This study sought to understand the plant host response against GLR at the molecular level and select useful genes for the breeding of resistant grapes. Hence, transcriptome analysis was conducted in two pathogen-inoculated Korean wild grape genotypes, namely, Vitis coignetiae Pulliant (Vc) and V. flexuosa Thunb (Vf), resistant and susceptible to GLR, respectively. RNAs were extracted from uninfected and infected leaves 24 h post-inoculation (hpi). In transcriptome analysis, a total of differentially expressed genes (DEGs) were 3979 and 2436 in the resistant and susceptible genotypes, of which 1765 and 1162 were up-regulated, and 2214 and 1274 were down-regulated, respectively. The top five up-regulated genes in Vc compared to Vf were homolog of carrot EP3-3 chitinase, calcineurin B-like proteins (CBL)-interacting protein kinase 20 (CIPK20), chalcone and stilbene synthase family protein, osmotin 34, and major latex protein (MLP)-like protein 423 (MLP423). The five most down-regulated genes in Vc compared to Vf were RADIALIS (RAD)-like 1, flavonol synthase 1, glutamine synthetase 2, RAD-like 6, and GDSL-like lipase/acylhydrolase superfamily protein, which were validated for their expression by real-time PCR. According to the Gene Ontology (GO) analysis, most of the up-regulated genes in Vc were associated with metabolic processes, whereas, most of down-regulated genes in Vf were associated with catalytic activity. The down-regulated genes in Vc and up-regulated genes in Vf were mostly associated with metabolic processes and catalytic activity. Defense-related genes, including phenylalanine ammonia-lyase (PAL), pathogenesis-related (PR) proteins, and salicylic acid (SA)-dependent responses were differentially expressed against the infection by the pathogen. This is the first report on the transcriptome analysis of grapevines in response to GLR and provides a basis for further studies on the discovery of resistant genes and the development of GLR-resistant grapevine varieties.