Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.