Infectious clones are important for functional studies of plant RNA viruses. Cowpea severe mosaic virus (CPSMV; Genus Comovirus, Family Comoviridae) is an RNA virus with a bipartite genome consisting of two single-stranded, plus-sense RNA segments. CPSMV is a well-known pathogen of cowpea, soybean, and other legume species. The RNA1 encodes proteins involved in replication, whereas the RNA2 encodes the movement protein and two coat proteins. In this study, we generated an infectious clone of a CPSMV variant able to infect tobacco (Nicotiana benthamiana) and soybean (Glycine max). The full-length cDNAs obtained from the CPSMV CB strain were cloned separately into the pAI101 binary vector downstream of a CaMV35S promoter with duplicated enhancers, and confirmed by restriction enzyme digestion and sequencing. Two amino acid changes were observed in the RNA1 polyprotein (S1098P and K1651R) when compared to the NCBI reference sequence (NC_003545.1), and none in the RNA2 polyprotein. The construct was introduced into lima bean cotyledons using particle bombardment. Inoculation with lima bean extracts resulted in systemic infection in both tobacco and soybean hosts. Infectivity was also retained throughout passages from soybean to tobacco. Surprisingly, Agrobacterium (C57C1) transformed with the construct harboring RNA1 cDNA grew into extremely small colonies on the selection plate, and did not grow at all in liquid culture without introducing mutations. Analysis of CPSMV mutants containing systematic deletions showed that the toxicity was imparted by a region in the N-terminal portion of the viral RNA-dependent RNA polymerase. This CPSMV infectious clone can be used as a plant virus vector to express foreign proteins or RNA, to study synergy between different comoviruses, and to study superinfection exclusion among bipartite viruses.