Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, and is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. Although the Korean BCoV vaccine strain, BC94, was isolated in 1995, there has still been no report of a molecular characterization of the vaccine strain. To characterize the vaccine strain, relationships between BC94 and field strains were investigated, based on sequence analysis and cross-immunity. We determined the complete sequences of the HE, N, and S genes from BC94 and four NVRQS isolates (SUN5, A3, 0501, 0502). Due to its major role in antigenicity, the spike proteins of the BCoVs were analyzed. BC94 showed distinctive genetic divergence from field isolates collected from 2002 to 2005. BC94, SUN5, and A3 had no virulence-specific sequence and there was a single amino acid change, from asparagine to lysine at residue 175, in the polymorphic region. Strains 0501 and 0502 had virulence-specific sequences at all seven sites. Although the recently isolated Korean BCoVs and BC94 were genetically different, the cleavage site of spike genes at 763~768 (KRRSRR) and the antigenic domain II of the spike protein, amino acid position 528, were conserved in all NVRQS isolates. The antigenic relatedness of KCD9, representative of recent Korean BCoVs, was compared with the Korean vaccine strain BC94. KCD9 showed cross-reactivity against BC94 by virus neutralization (VN) test. These results suggest that BC94 is antigenically closely related to field isolates and is still effective as a vaccine strain.