Single-stranded DNA extracted from virions of the originally described tomato yellow leaf curl virus (TYLCV) served as a template for in vitro synthesis of its double-stranded form. Analysis with different restriction enzymes yielded fragments with an overall size of approximately 5.6 kb, twice the suggested virus genome unit size. Based on restriction analysis and hybridization tests, it is suggested that the first described culture of TYLCV consists of two viral DNA populations reflecting some slight sequence differences. Agroinoculation of TYLCV hosts with one of the cloned viral DNA populations resulted in systemic infection, but symptoms on some tomato varieties were significantly milder than those induced by the native virus. Based on sequence data, the genomic organization of the mild infectious clone was found to be the same as described before for another Israeli isolate. However, the nucleotide sequence of its intergenic noncoding region, the putative replicase, and open reading frame C4 have only 78, 87, and 76% homology, respectively, compared with the formerly described Israeli severe TYLCV isolate.