Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively. ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms. SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis. These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N. benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation). SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity). Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region. Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N. benthamiana, emphasising their status as distinct virus species. In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious. Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV. When introduced into N. benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses. Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N. glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts. Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.