Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are structurally and genetically very similar. The specificity of the BMV and CMV coat proteins (CPs) during in vivo encapsidation was studied using two RNA3 chimera in which the respective CP genes were exchanged. The replicative competence of each chimera was analyzed in Nicotiana benthamiana protoplasts, and their ability to cause infections was examined in two common permissive hosts, Chenopodium quinoa and N. benthamiana. Each RNA3 chimera replicated to near wild-type (wt) levels and synthesized CPs of expected parental origin when co-inoculated with their respective genomic wt RNAs 1 and 2. However, inoculum containing each chimera was noninfectious in the common permissive hosts tested. Encapsidation assays in N. benthamiana protoplasts revealed that CMV CP expressed from chimeric BMV RNA3 was capable of packaging heterologous BMV RNA, however, at a lower efficiency than parental BMV CP. By contrast, BMV CP expressed from chimeric CMV RNA3 was unable to package heterologous CMV RNA. These observations demonstrate that BMV CP, but not CMV CP, exhibits a high degree of specificity during in vivo packaging. The reasons for the noninfectious nature of each chimera in the host plants tested and factors likely to affect encapsidation in vivo are discussed.