Begomoviruses (the family Geminiviridae) have either a monopartite or a bipartite (DNA A and DNA B) single-stranded DNA genome. DNA B contains the open reading frame for movement protein and enables some bipartite begomoviruses to invade non-phloem tissues, whereas monopartite begomoviruses are limited to the phloem. Betasatellite DNA replicates in association with some monopartite begomoviruses to produce severe symptoms and enhance replication of helper virus in some hosts. The cotton leaf curl Multan betasatellite (CLCuMuB) has been shown to substitute for the DNA B of a bipartite begomovirus by permitting systemic infection, but the tissue tropism and the role of betasatellites in releasing monopartite begomoviruses from the phloem to adjacent tissue has not been described. In this study, the tissue tropism of CLCuMuB and a monopartite helper virus, tomato leaf curl virus (ToLCV), has been investigated using in situ hybridization, promoter localization and analysis of transgenic 2β plants, which contain a dimer of the CLCuMuB genome. In situ hybridization showed that CLCuMuB and ToLCV were localized into the vascular tissue of infected plants. In addition, CLCuMuB was detected only in the vascular tissue of 2β transgenic plants after infection with ToLCV. β-glucuronidase (GUS) expression under the βC1 promoter was also limited to the phloem tissues. In conclusion, we showed for the first time that CLCuMuB localizes only in the phloem tissues and unlike the DNA B component, CLCuMuB is unable to release the monopartite helper virus out of phloem tissues.