Geminiviruses cause severe damage to agriculture worldwide. The replication (Rep) protein is the indispensable viral protein for viral replication. Although various functional domains of Rep protein in Geminivirus spp. have been characterized, the most carboxyl terminus of Rep protein was not available. We have reported the first cassava-infecting geminivirus, Sri Lankan cassava mosaic virus (SLCMV-HN7 strain), in China. In this study, we reported the second Chinese SLCMV strain, SLCMV-Col, and conducted comparative genomic analysis between these two SLCMV strains. The virulence of SLCMV-Col is much stronger than SLCMV-HN7, indicated by the higher virus titer, more severe symptoms, and more extent host defense. We functionally characterized that Rep protein, a 7-amino-acid motif at the most carboxyl terminus, is essential for Rep protein accumulation and virulence of SLCMV. We also provided evidence suggesting that the motif could also enhance triggering of salicylic acid (SA) defense against SLCMV infection in Nicotiana benthamiana. The significance of the balance between virulence and host SA defense responses in expanding invasions of SLCMV is also discussed.