Cysteine-rich receptor-like kinases (CRKs) are ubiquitous plant receptor-like kinases, which play a significant role in plant disease resistance. Gray mold is an economically important disease of grapes caused by Botrytis cinerea. However, CRK genes and their function in gray mold disease resistance in grapes have not been elucidated. This study aimed to identify and characterize CRKs in grapes and determine their role in gray mold resistance. Four CRKs were identified in Vitis amurensis and named VaCRK1–VaCRK4 according to their genomic distribution. The four VaCRKs were ectopically expressed in Arabidopsis thaliana to study their function in defense response against B. cinerea. Heterologous expression of VaCRK2 in A. thaliana conferred resistance to B. cinerea. VaCRK2 expression in gray mold-resistant grape cultivar increased significantly after B. cinerea inoculation and methyl jasmonate treatment. Furthermore, the expression of jasmonic acid (JA) signaling pathway-related genes in VaCRK2 overexpression lines of A. thaliana was significantly increased after B. cinerea inoculation, leading to the upregulation of pathogenesis-related (PR) genes and reactive oxygen species (ROS) accumulation. Overall, these results suggest that VaCRK2 confers resistance to B. cinerea by activating PR gene expression and oxidative burst through the JA signaling pathway.