The beet yellow stunt virus (BYSV) genome contains at least nine open reading frames (ORFs) that code for proteins ranging from 6 to 66 kDa. Based on amino acid sequence comparisons, the coat protein (CP) was previously identified as the product of ORF7. We expressed the product of ORF7 in bacteria and confirmed that ORF7 codes for the BYSV CP by immunoblotting. BYSV is a phloem-limited virus, and virus CP antigen of a quality sufficient for diagnostic antisera production has not been available. To produce BYSV antigen free of plant host contaminants, ORF7 was cloned into a pMAL bacterial expression vector. The resulting fusion protein was affinity-purified and used as an antigen to raise anti-BYSV CP antisera in rabbits and guinea pigs. Using these antisera, an indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA)-based diagnostic system was developed. This indirect DAS-ELISA format enabled reliable detection of BYSV in tissue extracts from virus-infected lettuce diluted up to 5,000 times. The diagnostic system developed may enable large-scale epidemiological studies of BYSV using simple serological techniques. The antisera raised had a titer exceeding 1 × 105 in immunoblots and easily detected the 23.7-kDa BYSV CP in virus-infected lettuce and sowthistle plants. In these two plant species, BYSV CP was detected as two closely migrating bands during electrophoresis, which may suggest posttranslational CP modifications. To further characterize the BYSV CP gene, the 5′-untranslated region (UTR) of the BYSV CP subgenomic RNA (sgRNA) was cloned and sequenced. The CP-encoding, approximately 1.9-kb sgRNA has an AT-rich, 66-nucleotide-long 5′-UTR colinear to the genomic sequence upstream of ORF7.