Geminiviruses are plant DNA viruses replicating by a rolling circle mechanism. We have investigated the specificity of replication origin recognition of two different isolates of tomato yellow leaf curl virus (TYLCV). Here, we show that TYLCV-Sardinian and -Israeli replication proteins display a high degree of specificity for their respective origins. The DNA sequences recognized are located on the left part of the intergenic region whereas the amino-terminal 116 amino acids of the Rep protein determine the specificity of origin recognition.